Antibodies that bind human CD27 and uses thereof

ABSTRACT

Isolated monoclonal antibodies which bind to human CD27 and related antibody-based compositions and molecules are disclosed. Also disclosed are therapeutic and diagnostic methods for using the antibodies.

RELATED APPLICATIONS

The present application is a divisional of U.S. patent application Ser.No. 13/086,286, filed Apr. 13, 2011, which claims priority to U.S.Provisional Application Nos. 61/323,720, filed Apr. 13, 2010; and61/471,459, filed on Apr. 4, 2011. The contents of the aforementionedapplications are hereby incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

Interactions between T cells and antigen-presenting cells involve avariety of accessory molecules that facilitate in the generation of animmune response. One such molecule is CD27, which binds CD70 and belongsto the tumor necrosis factor receptor (TNF-R) superfamily (Ranheim E A,et al., Blood. 1995 Jun. 15; 85(12):3556-65). CD27 typically exists as aglycosylated, type I transmembrane protein, frequently in the form ofhomodimers with a disulfide bridge linking the two monomers. Thedisulfide bridge is in the extracellular domain close to the membrane(Camerini et al., J Immunol. 147:3165-69 (1991). CD27 may also beexpressed in a soluble form (see, e.g., van Oers M H, et al., Blood.1993 Dec. 1; 82(11):3430-6 and Loenen W A, et al., Eur. J. Immunol.22:447, 1992). Cross-linking the CD27 antigen on T cells provides acostimulatory signal that, in concert with T-cell receptor crosslinking,can induce T-cell proliferation and cellular immune activation.

CD27 is expressed on mature thymocytes, on most CD4+ and CD8+ peripheralblood T cells, natural killer cells and B cells (Kobata T, et al., Proc.Natl. Acad. Sci. USA. 1995 Nov. 21; 92(24):11249-53). CD27 is alsohighly expressed on B cell non-Hodgkin's lymphomas and B cell chroniclymphocytic leukemias (Ranheim E A, et al., Blood. 1995 Jun. 15;85(12):3556-65). Additionally, increased levels of soluble CD27 proteinhave been identified in sera or sites of disease activity in parasiticinfection, cytomegalovirus (CMV) infection, sarcoidosis, multiplesclerosis, and B-cell chronic lymphocytic leukemia (Loenen W A, et al.,Eur. J. Immunol. 22:447, 1992).

Agonistic monoclonal antibodies against CD27 have recently been shown topromote T cell responses and show promise as anti-cancer therapeutics(see e.g., Sakanishi T, et al., Biochem Biophys. Res. Commun. 2010 Feb.18 and WO 2008/051424). However, while the results obtained to dateestablish CD27 as a useful target for immunotherapy, it is unknown whichparticular features of anti-CD27 monoclonal antibodies are especiallyadvantageous for therapeutic purposes. As such, there is a need in theart for further insight into the specific functional properties thatmake anti-CD27 antibodies therapeutically effective, as well as improvedtherapeutic antibodies against CD27 which are more effective fortreating and/or preventing diseases.

SUMMARY OF THE INVENTION

The present invention provides inter alia isolated anti-CD27 antibodieshaving particular functional properties which can be linked withadvantageous and desirable therapeutic effects. Specifically, anti-CD27monoclonal antibodies capable of up-regulating T cell mediated immuneresponses (e.g., as evidenced by inducement or enhancement ofantigen-specific T cell responses), which are particularly well-suitedfor combination with vaccine therapies, have been generated andcharacterized by way of the present invention. In one embodiment,agonist anti-CD27 antibodies can enhance the immune response againstcancers or infectious diseases by combination with active vaccination,or by enhancing endogenous immune responses. Such antibodies may alsodirectly or indirectly induce cytokine expression. Additionally,anti-CD27 antibodies that down-regulate T cell mediated immuneresponses, which are particularly well-suited for treating immunedisorders, such as graft rejection, allergy and autoimmune diseases,have been generated and characterized. Still further, anti-CD27antibodies that inhibit growth of CD27 expressing cells by direct cellkilling mechanisms (e.g., antibody dependent cell-mediated cytotoxicity(ADCC) and/or complement dependent cellular cytotoxicity (CDCC)), whichare particularly effective in treating a wide variety of diseasesinvolving cell proliferation (e.g., cancers), have been generated andcharacterized.

In one embodiment, the anti-CD27 antibodies of the present inventionexhibit one or more of the following properties:

(a) blocks binding of sCD70 to CD27 by at least about 70% at an antibodyconcentration of 10 μg/ml;

(b) binds to human CD27 with an equilibrium dissociation constant Kd of10⁻⁹ M or less, or alternatively, an equilibrium association constant Kaof 10⁺⁹ M⁻¹ or greater;

(c) induces specific complement mediated cytotoxicity (CDC) of CD27expressing cells of at least 10% at an antibody concentration of 3 μg/mland approximately 6% rabbit serum complement;

(d) induces antibody dependent cell-mediated cytotoxicity (ADCC)specific lysis of CD27 expressing cells of at least 10% at an antibodyconcentration of 3 μg/ml and ratio of effector:target cells of 75:1;

(e) improves median survival by at least 20% in severe combinedimmunodeficiency (SCID) mice post tumor cell inoculation in vivo (5×10⁵Raji cells or 1×10⁶ Daudi cells) when administered at 0.3 mg (i.p.) atleast twice a week for 3 weeks compared to mice to which antibody is notadministered;

(f) induces or enhances antigen-specific immune responses in combinationwith a vaccine or endogenous antigen;

(g) induces or enhances antigen-specific TH1 immune responses incombination with a vaccine or endogenous antigen;

(h) induces or enhances antigen-specific T-cell proliferation oractivation in combination with a vaccine or endogenous antigen;

(i) reduces or inhibits T-cell proliferation or activation;

(j) induces or enhances T-cell activity when combined with simultaneous,separate or sequential TCR activation;

(k) blocks binding of sCD70 to CD27 by at least about 70% at an antibodyconcentration of 10 μg/ml and reduces or inhibits T-cell activity whennot capable of binding to, or having reduced binding to Fc receptors;

(l) results in less than 50% depletion of CD3+ T-cells (other than NKcells) in macaques when administered at 3 mg/kg (i.v.) over the periodof 29 days immediately following administration; or

(m) results in less than 50% depletion of memory B-cells in macaqueswhen administered at 3 mg/kg (i.v.) over the period of 29 daysimmediately following administration.

In a particular embodiment, the antibodies of the invention exhibitcombinations of these functional properties.

Accordingly, in one aspect, the invention provides anti-CD27 antibodiesthat induce and/or enhance an immune response (e.g., a T cell mediatedimmune response). In a further embodiment, the antibodies inhibit thebinding of CD70 to CD27 on cells. Particular antibodies having thesecombinations of properties include mAb 1F5 comprising heavy and/or lightchain variable regions sequences comprising SEQ ID NOs: 37 and/or 43,respectively). Alternatively, the antibodies do not inhibit the bindingof CD70 to CD27 on cells. Particular antibodies having thesecombinations of properties include mAb 3H8 comprising heavy and/or lightchain variable regions sequences comprising SEQ ID NOs: 7 and/or 13,respectively, or 7 and/or 19, respectively). Such anti-CD27 antibodiesalso can be linked to a second molecule (e.g., as a bispecific molecule)having a binding specificity which is different from the antibody, suchas a T cell receptor (e.g., CD3, CD25, CD137, CD154), or an Fc receptor(e.g., FcγRI (CD64), FcγRIIA (CD32), FcγRIIB1 (CD32), FcγRIIB2 (CD32),FcγRIIIA (CD16a), FcγRIIIB (CD16b), FcεRI, FcεRII (CD23), FcαRI (CD89),Fcα/μR, and FcRn), or an NK receptor (e.g. CD56), or a B cell receptor(e.g. CD19, CD20).

Antibodies intended to be used for induction or enhancement of immuneresponses according to the present invention may have a functional Fcdomain permitting binding to Fc receptors, and may include a mutated Fcdomain having increased levels of binding to Fc receptors.

In another aspect, the invention provides anti-CD27 antibodies thatdown-regulate T cell mediated immune responses by inhibiting the bindingof CD27 to CD70 on cells which express these proteins. In a particularembodiment, the antibodies inhibit the binding of soluble CD70 (sCD70)to CD27 expressing cells by at least about 70%. Particular antibodiesfalling within this class include, e.g., mAb comprising heavy and/orlight chain variable regions sequences comprising SEQ ID NOs:37 and/or43 (mAb 1F5), SEQ ID NOs: 49 and/or 55 (mAb 1H8), or SEQ ID NOs: 103and/or 109 (mAb 3H12).

In yet another aspect, the invention provides anti-CD27 antibodies thatinduce or enhance effector cell function (e.g., cell killing via eitherADCC and/or CDC). In one embodiment, the antibody induces at least about30% specific lysis of CD27 expressing cells via ADCC at an antibodyconcentration of 10 μg/ml and/or induces at least about 30% CDC of CD27expressing cells at a concentration of 10 μg/ml. Particular antibodiesfalling within this class exhibiting ADCC effector function include,e.g., (e.g., mAb comprising heavy and/or light chain variable regionsequences comprising SEQ ID NOs: 61 and/or 67 (mAb 1G5), SEQ ID NOs: 85and/or 91, 85 and/or 97 (mAb 3A10), SEQ ID NOs:37 and/or 43 (mAb 1F5),SEQ ID NOs: 7 and/or 13, 7 and/or 19 (mAb 3H8), SEQ ID NOs: 49 and/or55(mAb 1H8), or SEQ ID NOs: 103 and/or 109 (mAb 3H12). In a furtherembodiment, the antibody also inhibits binding of CD70 to CD27 on cells.Particular antibodies having these combinations of functions include,e.g., (e.g., mAb comprising heavy and/or light chain variable regionssequences comprising SEQ ID NOs: 37 and/or 43(mAb 1F5), SEQ ID NOs: 49and/or 55 (mAb 1H8), SEQ ID NOs:103 and/or 109 (mAb 3H12).Alternatively, the antibody induces ADCC and/or CDC as described above,but does not inhibit binding of CD70 to CD27 on cells. Particularantibodies having these features include, e.g., mAb comprising heavyand/or light chain variable regions sequences comprising SEQ ID NOs: 61and/or 67 (mAb 1G5), SEQ ID NOs: 85 and/or 91, 85 and/or 97 (mAb 3A10),SEQ ID NOs:7 and/or 13, 7 and/or 19 (mAb 3H8). Anti-CD27 antibodiescapable of inducing or enhancing effector cell function (e.g., ADCCand/or CDC) can also be constructed to include an Fc region whichsuitably contributes a binding specificity for a specific Fc receptor(e.g., FcγRI (CD64), FcγRIIA (CD32), FcγRIIB1 (CD32), FcγRIIB2 (CD32),FcγRIIIA (CD16a), FcγRIIIB (CD16b), FcεRI, FcεRII (CD23), FcαRI (CD89),Fcα/μR, and FcRn).

In a further embodiment there is provided a method for enhancing animmune response against an antigen in a subject in need thereof byadministering to the subject: i) an anti-CD27 antibody and ii) anantigen, wherein the anti-CD27 antibody is administered separately fromand before the antigen is administered.

Typically in such a method the anti-CD27 antibody may be administeredbetween at least 2 and 96 hours before the antigen. For example, in sucha method, the anti-CD27 antibody may be administered at least 2 hoursbefore the antigen, for example at least 12 hours before the antigen,suitably at least 24 hours before the antigen, at least 48 hours beforethe antigen or at least 72 hours before the antigen.

wherein the TLR agonist is a TLR3 agonist.

In a further embodiment there is provided a method for enhancing animmune response against an antigen in a subject in need thereof bysimultaneously, separately or sequentially administering to the subject:i) an anti-CD27 antibody; ii) a TLR agonist; and iii) optionally, theantigen.

In a preferred embodiment of such a method the TLR agonist is a TLR3agonist, for example but not limited to Poly IC:LC.

CD27 expressing cells include any and all cells that express CD27,including, but not limited to B cells, NK cells and T cells. In aparticular embodiment, the CD27 expressing cells include cancer celllines such as Jurkat cells, Raji cells, Ramos cells and Daudi cells. Inanother embodiment, the CD27 expressing cells are tumor cells or cancercells. In another embodiment, CD27 expressing cells include B cells, NKcells, and T cells including T cells that are found infiltrating tumors,also called tumor infiltrating lymphocytes.

Particular antibodies of the invention comprise heavy and light chainvariable regions that utilize particular human germlines, i.e., areencoded by the germline genes, but include genetic rearrangements andmutations, e.g., somatic mutations, which occur during antibodymaturation. In one embodiment, the heavy chain variable region of theantibodies of the present invention is derived from a human germline 3-7or 3-33 gene. In another embodiment, the light chain variable region ofthe antibody is derived from a human germline 3-20, 3-11, 24, 1D-16, or1-13 gene. In a particular embodiment, the heavy chain variable regionof the antibody is derived from a human germline V_(H) 3-7 or V_(H) 3-33gene and the light chain variable region of the antibody is derived froma human germline V_(K) 3-20, V_(K) 3-11, V_(K) 1 D-16, or V_(K) 1-13gene.

A V_(H) 3-33 germline sequence is provided (Genbank Accession NoAAP44382) as follows:

(SEQ ID NO: 3) 1vqlvesgggv vqpgrslrls caasgftfst ygmhwvrqap gkglewvaii wfdgsntyya 61dsvrgrftis rdssrktlyl emkslrvedt avyycakA V_(H) 3-7 germline sequence is provided (Genbank Accession NoAAP44389) as follows:

(SEQ ID NO: 4) 1vqlvesgggl vqpggslrls caasgftfsn symtwvrqap gkglewvani kpdgsdknyi 61nsvrgrftis rdnaekssyl qmnslraedt aiyycvt

In another embodiment, the heavy chain variable region CDR3 sequence isselected from the group consisting of SEQ ID NOs: 10, 28, 40, 52, 64,76, 88, 106, and conservative sequence modifications thereof (e.g.,conservative amino acid substitutions). The antibodies may furtherinclude a light chain variable region CDR3 sequence selected from thegroup consisting of SEQ ID NOs: 16, 22, 34, 46, 58, 70, 82, 94, 100,112, and conservative sequence modifications thereof. In anotherembodiment, the heavy chain CDR2 and CDR1 sequences are selected fromSEQ ID NOs: 9, 27, 39, 51, 63, 75, 87, 105, and SEQ ID NOs: 8, 26, 38,50, 62, 74, 86, 104, respectively, and conservative sequencemodifications thereof. The light chain CDR2 and CDR1 sequences areselected from SEQ ID NOs: 15, 21, 33, 45, 57, 69, 81, 93, 99, 111, andSEQ ID NOs: 14, 20, 32, 44, 56, 68, 80, 92, 98, 110, respectively, andconservative sequence modifications thereof.

In still another embodiment, the invention provides an isolated antibodythat binds CD27 and includes heavy and light chain variable region CDR1,CDR2 and CDR3 sequences selected from the group consisting of:

-   -   (i) a heavy chain variable region CDR1 comprising SEQ ID NO: 38;        -   a heavy chain variable region CDR2 comprising SEQ ID NO: 39;        -   a heavy chain variable region CDR3 comprising SEQ ID NO: 40;        -   a light chain variable region CDR1 comprising SEQ ID NO: 44;        -   a light chain variable region CDR2 comprising SEQ ID NO: 45;        -   a light chain variable region CDR3 comprising SEQ ID NO: 46;            or        -   conservative sequence modifications thereof;    -   (ii) a heavy chain variable region CDR1 comprising SEQ ID NO:        50;        -   a heavy chain variable region CDR2 comprising SEQ ID NO: 51;        -   a heavy chain variable region CDR3 comprising SEQ ID NO: 52;        -   a light chain variable region CDR1 comprising SEQ ID NO: 56;        -   a light chain variable region CDR2 comprising SEQ ID NO: 57;        -   a light chain variable region CDR3 comprising SEQ ID NO: 58;            or        -   conservative sequence modifications thereof;    -   (iii) a heavy chain variable region CDR1 comprising SEQ ID NO:        104;        -   a heavy chain variable region CDR2 comprising SEQ ID NO:            105;        -   a heavy chain variable region CDR3 comprising SEQ ID NO:            106;        -   a light chain variable region CDR1 comprising SEQ ID NO:            110;        -   a light chain variable region CDR2 comprising SEQ ID NO:            111;        -   a light chain variable region CDR3 comprising SEQ ID NO:            112; or        -   conservative sequence modifications thereof;    -   (iv) a heavy chain variable region CDR1 comprising SEQ ID NO:        86;        -   a heavy chain variable region CDR2 comprising SEQ ID NO: 87;        -   a heavy chain variable region CDR3 comprising SEQ ID NO: 88;        -   a light chain variable region CDR1 comprising SEQ ID NO: 92            or 98;        -   a light chain variable region CDR2 comprising SEQ ID NO: 93            or 99;        -   a light chain variable region CDR3 comprising SEQ ID NO: 94            or 100;        -   or conservative sequence modifications thereof;    -   (v) a heavy chain variable region CDR1 comprising SEQ ID NO: 26;        -   a heavy chain variable region CDR2 comprising SEQ ID NO: 27;        -   a heavy chain variable region CDR3 comprising SEQ ID NO: 28;        -   a light chain variable region CDR1 comprising SEQ ID NO: 32;        -   a light chain variable region CDR2 comprising SEQ ID NO: 33;        -   a light chain variable region CDR3 comprising SEQ ID NO: 34;            or        -   conservative sequence modifications thereof;    -   (vi) a heavy chain variable region CDR1 comprising SEQ ID NO:        74;        -   a heavy chain variable region CDR2 comprising SEQ ID NO: 75;        -   a heavy chain variable region CDR3 comprising SEQ ID NO: 76;        -   a light chain variable region CDR1 comprising SEQ ID NO: 80;        -   a light chain variable region CDR2 comprising SEQ ID NO: 81;        -   a light chain variable region CDR3 comprising SEQ ID NO: 82;            or        -   conservative sequence modifications thereof;    -   (vii) a heavy chain variable region CDR1 comprising SEQ ID NO:        8;        -   a heavy chain variable region CDR2 comprising SEQ ID NO: 9;        -   a heavy chain variable region CDR3 comprising SEQ ID NO: 10;        -   a light chain variable region CDR1 comprising SEQ ID NO: 14            or 20;        -   a light chain variable region CDR2 comprising SEQ ID NO: 15            or 21;        -   a light chain variable region CDR3 comprising SEQ ID NO: 16            or 22;        -   or conservative sequence modifications thereof; and    -   (viii) a heavy chain variable region CDR1 comprising SEQ ID NO:        62;        -   a heavy chain variable region CDR2 comprising SEQ ID NO: 63;        -   a heavy chain variable region CDR3 comprising SEQ ID NO: 64;        -   a light chain variable region CDR1 comprising SEQ ID NO: 68;        -   a light chain variable region CDR2 comprising SEQ ID NO: 69;        -   a light chain variable region CDR3 comprising SEQ ID NO: 70;            or        -   conservative sequence modifications thereof.

In another embodiment, the heavy chain variable region CDR3 sequencecomprises an amino acid sequence selected from the consensus sequence: R(G,E,D) (S,L,G,-) (G,L,T,W,-) (N,A,T,H,-) (V,T,-) (M,P,-) (G,V,-) (R,-)(G,M,-) (D,H,L,T,W) (A,G,N,W) (D,F,V,Y) (F,L) (D,E) (H,I,L,Y) (SEQ IDNO: 113), wherein “-” denotes the option of no amino acid residue beingpresent at that consensus position. The antibodies may further include alight chain variable region CDR3 sequence comprising an amino acidsequence selected from the consensus sequence: Q (F,R,Y) (N,S) (N,T,S)(Y,W) P (F,L,P,R) T (SEQ ID NO: 114), wherein “-” denotes the option ofno amino acid residue being present at that consensus position. Inanother embodiment, the heavy chain variable region CDR2 sequencecomprises an amino acid sequence selected from the consensus sequence: I(K,W) (Y,N,Q) D G S (E,N) (K,Q) (SEQ ID NO: 115), wherein “-” denotesthe option of no amino acid residue being present at that consensusposition, and the light chain variable region CDR2 sequence comprises anamino acid sequence selected from the consensus sequence: (A,D) A S (SEQID NO: 116). In another embodiment, the heavy chain variable region CDR1sequence comprises an amino acid sequence selected from the consensussequence: G F (T,S) (F,L) (S,N) (I,S,H) (Y,H) (SEQ ID NO: 117); and thelight chain variable region CDR1 sequence comprises an amino acidsequence selected from the consensus sequence: Q (D,G,S) (I,V) (D,S)(R,S) (A,W,Y) (SEQ ID NO: 118).

In another embodiment, isolated antibodies of the invention bind tohuman CD27 and include a heavy chain variable region including an aminoacid sequence selected from the group consisting of SEQ ID NOs: 6, 7,24, 25, 36, 37, 48, 49, 60, 61, 72, 73, 84, 85, 102, 103, andconservative sequence modifications thereof. The antibody may furtherinclude a light chain variable region including an amino acid sequenceselected from the group consisting of SEQ ID NOs: 12, 13, 18, 19, 30,31, 42, 43, 54, 55, 66, 67, 78, 79, 90, 91, 96, 97, 108, 109, andconservative sequence modifications thereof.

In a still further embodiment, isolated antibodies of the invention bindto human CD27 and include a heavy chain variable region and a lightchain variable region including the amino acid sequences selected fromthe group consisting of:

(a) SEQ ID NOs: 37 and/or 43, respectively, and conservative sequencemodifications thereof;

(b) SEQ ID NOs: 49 and/or 55, respectively, and conservative sequencemodifications thereof;

(c) SEQ ID NOs: 103 and/or 109, respectively, and conservative sequencemodifications thereof;

(d) SEQ ID NOs: 85 and/or 91 and/or 97, respectively, and conservativesequence modifications thereof;

(e) SEQ ID NOs: 25 and/or 31, respectively, and conservative sequencemodifications thereof;

(f) SEQ ID NOs: 73 and/or 79, respectively, and conservative sequencemodifications thereof;

(g) SEQ ID NOs: 7 and/or 13 and/or 19, respectively, and conservativesequence modifications thereof; and

-   -   (h) SEQ ID NOs: 61 and/or 67, respectively, and conservative        sequence modifications thereof;

Isolated antibodies which include heavy and light chain variable regionshaving at least 80%, or at least 85%, or at least 90%, or at least 95%,or at least 96%, or at least 97%, or at least 98%, or at least 99%, ormore sequence identity to any of the above sequences are also includedin the present invention. Ranges intermediate to the above-recitedvalues, e.g., heavy and light chain variable regions having at least80-85%, 85-90%, 90-95% or 95-100% sequence identity to any of the abovesequences are also intended to be encompassed by the present invention.

Also encompassed by the present invention are isolated antibodies whichcompete for binding to CD27 with the antibodies of the invention. In aparticular embodiment, the antibody competes for binding to CD27 with anantibody comprising heavy and/or light chain variable regions comprisingthe amino acid sequences set forth in SEQ ID NOs: 37 and 43, SEQ ID NOs:49 and 55, SEQ ID NOs: 103 and 109, SEQ ID NOs: 85 and 91, SEQ ID NOs:85 and 97, SEQ ID NOs: 25 and 31, SEQ ID NOs: 73 and 79, SEQ ID NOs: 7and 13, SEQ ID NOs: 7 and 19, SEQ ID NOs: 61 an 67, respectively, oramino acid sequences at least 80% identical thereto. In anotherembodiment, the antibody competes for binding to CD27 with an antibodycomprising heavy and/or light chain variable regions comprising theamino acid sequences set forth in SEQ ID NOs: 37 and 43 (1F5), SEQ IDNOs: 49 and 55 (1H8) or SEQ ID NOs: 103 and 109 (3H12). In anotherembodiment, the antibody competes for binding to CD27 with an antibodycomprising heavy and/or light chain variable regions comprising theamino acid sequences set forth in SEQ ID NOs: 25 and 31 (2C2), SEQ IDNOs: 7 and 13 (3H8), SEQ ID NOs: 7 and 19 (3H8), SEQ ID NOs: 61 an 67(1G5) or SEQ ID NOs: 73 and 79 (2G9). In yet another embodiment, theantibody competes for binding to CD27 with an antibody comprising heavyand/or light chain variable regions comprising the amino acid sequencesset forth in SEQ ID NOs: 85 and 91 (3A10) or SEQ ID NOs: 85 and 97(3A10).

Other antibodies of the invention bind to an epitope on CD27 recognizedby the antibodies described herein. In another particular embodiment,the antibody binds to an epitope on CD27 recognized by an antibodycomprising heavy and/or light chain variable regions comprising theamino acid sequences set forth in SEQ ID NOs: 37 and 43, SEQ ID NOs: 49and 55, SEQ ID NOs: 103 and 109, SEQ ID NOs: 85 and 91, SEQ ID NOs: 85and 97, SEQ ID NOs: 25 and 31, SEQ ID NOs: 73 and 79, SEQ ID NOs: 7 and13, SEQ ID NOs: 7 and 19, SEQ ID NOs: 61 an 67, respectively, or aminoacid sequences at least 80% identical thereto. In another embodiment,the antibody binds to an epitope on CD27 recognized by an antibodycomprising heavy and/or light chain variable regions comprising theamino acid sequences set forth in SEQ ID NOs: 37 and 43 (1F5), SEQ IDNOs: 49 and 55 (1H8) or SEQ ID NOs: 103 and 109 (3H12). In anotherembodiment, the antibody binds to an epitope on CD27 recognized by anantibody comprising heavy and/or light chain variable regions comprisingthe amino acid sequences set forth in SEQ ID NOs: 25 and 31 (2C2), SEQID NOs: 7 and 13 (3H8), SEQ ID NOs: 7 and 19 (3H8), SEQ ID NOs: 61 an 67(1G5) or SEQ ID NOs: 73 and 79 (2G9). In yet another embodiment, theantibody binds to an epitope on CD27 recognized by an antibodycomprising heavy and/or light chain variable regions comprising theamino acid sequences set forth in SEQ ID NOs: 85 and 91 (3A10) or SEQ IDNOs: 85 and 97 (3A10).

The antibodies of the invention can either be full-length, for example,any of the following isotypes: IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2,IgAsec, IgD, and IgE. Alternatively, the antibodies can be fragmentssuch as an antigen-binding portion or a single chain antibody (e.g., aFab, F(ab′)₂, Fv, a single chain Fv fragment, an isolatedcomplementarity determining region (CDR) or a combination of two or moreisolated CDRs). The antibodies can be any kind of antibody, including,but not limited to, human, humanized, and chimeric antibodies.

Tumor antigens employed by the present invention (e.g., in a vaccine,used in combination with an anti-CD27 antibody of the invention) includeany antigen or antigenic determinant which is present on (or associatedwith) a tumor cell and not typically on normal cells, or an antigen orantigenic determinant which is present on or associated with tumor cellsin greater amounts than on normal (non-tumor) cells, or an antigen orantigenic determinant which is present on tumor cells in a differentform than that found on normal (non-tumor) cells. Such antigens includetumor-specific antigens, including tumor-specific membrane antigens,tumor-associated antigens, including tumor-associated membrane antigens,embryonic antigens on tumors, growth factor receptors, growth factorligands, and any other type of antigen that is associated with cancer. Atumor antigen may be, for example, an epithelial cancer antigen, (e.g.,breast, gastrointestinal, lung), a prostate specific cancer antigen(PSA) or prostate specific membrane antigen (PSMA), a bladder cancerantigen, a lung (e.g., small cell lung) cancer antigen, a colon cancerantigen, an ovarian cancer antigen, a brain cancer antigen, a gastriccancer antigen, a renal cell carcinoma antigen, a pancreatic cancerantigen, a liver cancer antigen, an esophageal cancer antigen, a headand neck cancer antigen, or a colorectal cancer antigen. For example,the antigen may include a tumor antigen, such as βhCG, gp100 or Pmel17,CEA, gp100, TRP-2, NY-BR-1, NY-CO-58, MN (gp250), idiotype, Tyrosinase,Telomerase, SSX2, MUC-1, MAGE-A3, and high molecular weight-melanomaassociated antigen (HMW-MAA) MART1, melan-A, EGFRvIII, NY-ESO-1, MAGE-1,MAGE-3, WT1, Her2, or mesothelin. Other antigens employed by the presentinvention (e.g., in a vaccine, used in combination with an anti-CD27antibody of the invention) include antigens from infectious diseasepathogens, such as viruses, bacteria, parasites and fungi, examples ofwhich are disclosed herein.

The invention also provides a bispecific molecule comprising an antibodyof the invention linked to a second functional moiety having a differentbinding specificity than said antibody. For example, in one embodiment,the second molecule may bind a T cell receptor (e.g., CD3, CD40).

Compositions including an antibody or a bispecific molecule describedherein, formulated with a pharmaceutically acceptable carrier, are alsoprovided. The compositions may further include an adjuvant,immunostimulatory agent (e.g., CD40 ligand, FLT 3 ligand, cytokines,colony-stimulating factors, an anti-CTLA-4 antibody, anti-PD1 antibody,anti-41BB antibody, anti OX-40 antibody, LPS (endotoxin), ssRNA, dsRNA,Bacille Calmette-Guerin (BCG), Levamisole hydrochloride, intravenousimmune globulins and a Toll-like Receptor (TLR) agonist (e.g., TLR3agonist such as Poly IC, a TLR4 agonist, a TLR5 agonist, a TLR7 agonist,a TLR8 agonist, and a TLR 9 agonist)), immunosuppressive agent, anotherantibody, or an antigen. Exemplary antigens include, but are not limitedto, a component of a pathogen, a tumor antigen (e.g., βhCG, gp100 orPmel17, HER2/neu, WT1, mesothelin, CEA, gp100, MART1, TRP-2, melan-A,NY-ESO-1, NY-BR-1, NY-CO-58, MN (gp250), idiotype, MAGE-1, MAGE-3,MAGE-A3, Tyrosinase, Telomerase, SSX2 antigens, MUC-1 antigens, and germcell derived tumor antigens), an infectious disease antigen (e.g. viralantigens, bacterial and parasitic antigens) an allergen, or anautoantigen. Any of the antigens disclosed herein can be included in acomposition of the invention.

Nucleic acid molecules encoding the antibodies of the invention are alsoencompassed by the invention, as well as expression vectors comprisingsuch nucleic acids and host cells comprising such expression vectors.For example, in one embodiment, the invention provides an isolatedmonoclonal antibody that binds human CD27, wherein the antibodycomprises a heavy chain variable region and a light chain variableregion encoded by nucleic acid sequences selected from the groupconsisting of: (a) SEQ ID NOs: 5 and 11, respectively; (b) SEQ ID NOs: 5and 17, respectively; (c) SEQ ID NOs: 23 and 29, respectively; (d) SEQID NOs: 35 and 41, respectively; (e) SEQ ID NOs: 47 and 53,respectively; (f) SEQ ID NOs: 59 and 65, respectively; (g) SEQ ID NOs:71 and 77, respectively; (h) SEQ ID NOs: 83 and 89, (i) SEQ ID NOs: 83and 95; (j) SEQ ID NOs: 101 and 107, respectively or nucleic acidsequences having at least 90% identity to the nucleic acid sequences of(a)-(h).

In another embodiment, the present invention provides methods forinducing or enhancing an immune response (e.g., a T cell-mediated immuneresponse, and/or an NK-mediated response and/or a B cell-mediated immuneresponse) against an antigen in a subject by administering to thesubject an effective amount of an antibody (e.g., a full lengthantibody), composition or bispecific molecule described herein. Suchmethods are particularly well-suited for use in vaccine therapies.

The antibodies and other compositions of the present invention can alsobe used to inhibit growth of CD27 expressing cells by contacting thecells with an antibody or composition in an amount effective to inhibitgrowth of CD27 expressing cells (e.g., in the treatment of cancers).Antibodies useful in inhibiting the growth of CD27 expressing cellsinclude full length antibodies and fragments thereof, as well asantibodies that contain a second binding specificity for an Fc receptor.In one embodiment, the CD27 expressing cells are contacted with anantibody in the presence of effector cells under conditions sufficientto induce antibody-dependent cellular cytoxicity (ADCC) of target cells(e.g., the antibody induces at least about 40% specific lysis of CD27expressing cells at a concentration of 10 μg/ml and comprises SEQ IDNOs:61, 67, 85, 91, 97, 37, and/or 43). In another embodiment, the cellsare contacted with an antibody under conditions sufficient to inducecomplement mediated cytotoxicity (CDC) of the cells (e.g., the antibodyinduces at least about 40% complement mediated cytotoxicity (CDC) ofCD27 expressing cells at a concentration of 10 μg/ml and comprises SEQID NOs: 7, 13, 19, 49, 55, 103, and/or 109).

In a further embodiment, the antibody employed to inhibit growth of CD27expressing cells may also possess (or lack) additional functionalfeatures. For example, the antibody may also inhibit the binding of CD70to CD27 on cells which express these proteins (e.g., a mAb comprisingheavy and/or light chain variable regions sequences comprising SEQ IDNOs: 37 and/or 43 (mAb 1F5), SEQ ID NOs: 49 and/or 55 (mAb 1H8), or SEQID NOs: 103 and/or 109 (mAb 3H12). Alternatively, the antibody may notinhibit the binding of CD70 to CD27 on such cells (e.g., a mAbcomprising heavy and/or light chain variable regions sequencescomprising SEQ ID NOs: 61 and/or 67 (mAb 1G5), SEQ ID NOs: 85 and/or 91,85 and/or 97 (mAb 3A10), or SEQ ID NOs:7 and/or 13, 7 and/or 19 (mAb3H8).

CD27 expressing cells include any and all cells the express CD27,including, but not limited to NK cells, B cells and T cells. In aparticular embodiment, the CD27 expressing cells include cell lines suchas Jurkat cells, Raji cells, Ramos cells and Daudi cells. In anotherembodiment, the CD27 expressing cells are tumor cells or cancer cells.In another embodiment, CD27 expressing cells include B cells, NK cells,T cells that are found infiltrating tumor or cancer cells, also calledtumor infiltrating lymphocytes.

The methods of inhibiting the growth of CD27 expressing cells that aredescribed herein can be used to treat and prevent a wide variety ofdiseases and disorders. For example, in one embodiment, the methods canbe used to treat or prevent a cancer (e.g., a cancer selected from thegroup consisting of leukemia, acute lymphocytic leukemia, acutemyelocytic leukemia, myeloblasts promyelocyte myelomonocytic monocyticerythroleukemia, chronic leukemia, chronic myelocytic (granulocytic)leukemia, chronic lymphocytic leukemia, mantle cell lymphoma, primarycentral nervous system lymphoma, Burkitt's lymphoma, marginal zone Bcell lymphoma, Polycythemia vera Lymphoma, Hodgkin's disease,non-Hodgkin's disease, multiple myeloma, Waldenstrom'smacroglobulinemia, heavy chain disease, solid tumors, sarcomas, andcarcinomas, fibrosarcoma, myxosarcoma, liposarcoma, chrondrosarcoma,osteogenic sarcoma, osteosarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon sarcoma, colorectal carcinoma, pancreaticcancer, breast cancer, ovarian cancer, prostate cancer, squamous cellcarcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma,sebaceous gland carcinoma, papillary carcinoma, papillaryadenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogeniccarcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma,choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervicalcancer, uterine cancer, testicular tumor, lung carcinoma, small celllung carcinoma, non small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma,retinoblastoma, nasopharyngeal carcinoma, esophageal carcinoma, basalcell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brainand central nervous system (CNS) cancer, cervical cancer,choriocarcinoma, colorectal cancers, connective tissue cancer, cancer ofthe digestive system, endometrial cancer, esophageal cancer, eye cancer,head and neck cancer, gastric cancer, intraepithelial neoplasm, kidneycancer, larynx cancer, liver cancer, lung cancer (small cell, largecell), melanoma, neuroblastoma; oral cavity cancer (for example lip,tongue, mouth and pharynx), ovarian cancer, pancreatic cancer,retinoblastoma, rhabdomyosarcoma, rectal cancer; cancer of therespiratory system, sarcoma, skin cancer, stomach cancer, testicularcancer, thyroid cancer, uterine cancer, and cancer of the urinarysystem). Preferred cancers include CD27-expressing tumors selected fromthe group consisting of chronic lymphocytic leukemia, mantle celllymphoma, primary central nervous system lymphoma, Burkitt's lymphomaand marginal zone B cell lymphoma. In another embodiment, the methodscan be used to treat or prevent a bacterial, fungal, viral or parasiticinfection.

The present invention further provides methods for inhibiting thebinding of CD70 to CD27 on cells in a subject having a disorder byadministering to the subject antibodies or compositions as describedherein, as well as methods for down-regulating a T cell response in anindividual having a disorder by administering to a subject antibodies orcompositions described herein. These methods are ideally suited for usein the treatment of immune disorders, such as graft rejection,autoimmune diseases, and allergy. Antibodies useful in these methodsinclude Fab fragments, as well as a mutated Fc region so that theantibody does not bind, or has significantly reduced binding to, Fcreceptors. In a particular embodiment, the antibody comprises heavyand/or light chain variable regions sequences comprising SEQ ID NOs: 37and/or 43 (mAb 1F5), SEQ ID NOs: 49 and/or 55 (mAb 1H8), or SEQ ID NOs:103 and/or 109 (mAb 3H12).

The methods described herein for inhibiting the binding of CD70 to CD27on cells and for down-regulating a T cell response can be used to treata wide variety of diseases and disorders, including, but not limited tograft rejection, allergy and autoimmune diseases. In a particularembodiment, the disease is an autoimmune disease (e.g., multiplesclerosis, rheumatoid arthritis, type 1 diabetes, psoriasis, Crohn'sdisease and other inflammatory bowel diseases such as ulcerativecolitis, systemic lupus eythematosus (SLE), autoimmuneencephalomyelitis, myasthenia gravis (MG), Hashimoto's thyroiditis,Goodpasture's syndrome, pemphigus, Graves disease, autoimmune hemolyticanemia, autoimmune thrombocytopenic purpura, scleroderma withanti-collagen antibodies, mixed connective tissue disease, polypyositis,pernicious anemia, idiopathic Addison's disease, autoimmune associatedinfertility, glomerulonephritis, crescentic glomerulonephritis,proliferative glomerulonephritis, bullous pemphigoid, Sjogren'ssyndrome, psoriatic arthritis, insulin resistance, autoimmune diabetesmellitus, autoimmune hepatitis, autoimmune hemophilia, autoimmunelymphoproliferative syndrome (ALPS), autoimmune hepatitis, autoimmunehemophilia, autoimmune lymphoproliferative syndrome, autoimmuneuveoretinitis, Guillain-Bare syndrome, arteriosclerosis and Alzheimer'sdisease).

The present invention further provides for particular uses for theantibodies, compositions and bispecific molecules described herein. Forexample, in one embodiment, the invention provides for the use of anantibody, composition or bispecific molecule in the manufacture of amedicament for inducing or enhancing an immune response against anantigen in a subject. In further embodiments, the invention provides forthe use of an antibody or composition in the manufacture of a medicamentfor inhibiting growth of CD27 expressing cells, the use of an antibodyor composition in the manufacture of a medicament for inhibiting thebinding of CD70 to CD27 on cells in a subject having a disorder, and theuse of an antibody or composition in the manufacture of a medicament fordown-regulating a T cell response in an individual having a disorder.The present invention further includes an antibody, composition orbispecific molecule for use in inducing or enhancing an immune responseagainst an antigen in a subject, an antibody or composition for use ininhibiting growth of CD27 expressing cells, an antibody or compositionfor use in inhibiting the binding of CD70 to CD27 on cells in a subjecthaving a disorder, and an antibody or composition for use indown-regulating a T cell response in an individual having a disorder.

The present invention also provides methods for detecting the presenceor absence of CD27 in a biological sample by (1) contacting a biologicalsample with an antibody described herein (wherein the antibody islabeled with a detectable substance) and (2) detecting the antibodybound to CD27.

Also within the scope of the invention are kits comprising thecompositions (e.g., antibodies and/or bispecific molecules) of theinvention and, optionally, instructions for use. The kit can furthercontain a least one additional reagent, such as a cytokine orcomplement, or one or more additional antibodies of the invention.

Other features and advantages of the instant invention will be apparentfrom the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the affinity and kinetic parameters for mAbs 1G5, 1H8,3H12, 3H8, 2G9, 1F5, 3A10, 2C2, ms 1A4, ms 9F4 and ms M-T271 asdetermined by Biacore™ BiaEvaluation software (Biacore AB) withrecombinant human CD27 immobilized on the chip.

FIG. 2 is a graph showing the binding of human anti-CD27 antibodies(2C2, 3H8, 1F5, 1G5, 1H8, 2G9, 3A10 and 3H12) to recombinant purifiedhuman CD27 using ELISA.

FIG. 3 is a graph showing binding by ELISA of 1F5 to purifiedrecombinant human or monkey (macaque) CD27.

FIG. 4 is a graph depicting the effect of human anti-CD27 antibodies(2C2, 3H8, 1F5, 1G5, 1H8, 2G9, 3A10 and 3H12) and MsIgG's (1A4, 9F4, andM-T271) on the binding of soluble CD70 (sCD70) to CD27 protein (shown as% blocking) by ELISA.

FIG. 5 is a flow cytometric analysis of 1F5 binding to humanlymphoblastoid cell lines, and blocking of sCD70 binding.

FIGS. 6A-D are graphs showing the binding of human anti-CD27 antibodies(2C2, 3H8, and 1F5) to CD27 on Jurkat cells (FIG. 4A), Raji cells (FIG.4B), Ramos cells (FIG. 4C), and Daudi cells (FIG. 4D) as assessed byflow cytometry.

FIG. 7 is a graph showing the binding of human anti-CD27 antibodies(2C2, 3H8, 1F5, 1G5, 1H8, 2G9, 3A10 and 3H12) to CD27 on Daudi cells asassessed by flow cytometry.

FIG. 8 is a bar graph showing the results of an anti-CD27 cross-blockingELISA experiment, demonstrating that antibodies 1F5, 1H8 and 3H12 arecapable of cross-blocking each other and thus bind to the same epitope.

FIG. 9 is a bar graph showing the results of an anti-CD27 cross-blockingELISA experiment, demonstrating that antibodies 2C2, 3H8, 1G5 and 2G9are capable of cross-blocking each other and thus bind to the sameepitope.

FIG. 10 is a bar graph showing the results of an anti-CD27cross-blocking ELISA experiment, demonstrating that the binding ofantibody 3A10 to CD27 is not fully cross-blocked by any of the otheranti-CD27 antibodies tested, thus indicating that 3A10 binds a uniqueepitope, but 3A10 binding is partially cross-blocked by antibodies 1F5,1H8 and 3H12, indicating that the epitope for 3A10 may be close to theepitope bound by antibodies 1F5, 1H8 and 3H12.

FIG. 11 is a graph depicting the results of a complement dependentcellular cytotoxicity (CDCC) assay using mAbs 1F5, 2C2, 3H8, 1G5, 1H8,2G9, 3A10 and 3H12.

FIG. 12 is a graph depicting the results of a further complementdependent cellular cytotoxicity (CDCC) assay using mAb 1F5

FIG. 13 is a graph depicting the results of an antibody dependentcell-mediated cytotoxicity (ADCC) assay using mAbs 2C2, 1F5, 3H8, 1G5,1H8, 2G9, 3A10, 3H12, Rituxan and HuIgG.

FIG. 14 is a graph depicting the results of a further antibody dependentcell-mediated cytotoxicity (ADCC) using mAb 1F5

FIG. 15 is an alignment of the VH sequences of human anti-CD27antibodies (1F5, 1G5, 1H8, 2C2, 2G9, 3A10, 3H12 and 3H8).

FIG. 16 is an alignment of the VL sequences of human anti-CD27antibodies (1F5, 1G5, 1H8, 2C2, 2G9, 3A10, 3H12 and 3H8).

FIGS. 17 and 18 show results from an in vivo non-human primate studyusing mAb 1F5. In particular, FIG. 17 shows 1F5 on circulatinglymphocytes after a single dose. FIG. 18 shows 1F5 does notsignificantly deplete circulating lymphocytes.

FIG. 19 depicts the results of a pentamer staining assay on mouseperipheral blood cells and splenocytes.

FIG. 20 depicts the results of an ELISPOT assay and enhanced IFN′production using anti-CD27 antibodies.

FIG. 21 shows enhancement by anti-CD27 mAbs of T cell responses to avaccine antigen in a transgenic mouse model by pentamer staining and IFNELISPOT.

FIGS. 22A-C are the protocol for and results of an experiment showingthat anti-CD27 enhances T cell responses to an APC-targeted vaccine(α-DEC205-OVA). FIG. 22A shows the protocol for the experiment. FIG. 22Bshows the results of a tetramer staining experiment to measureantigen-specific T cells. FIG. 22C shows the results of an IFN-gammaELISPOT assay to measure antigen-specific T cells.

FIGS. 23A-D are the results of an experiment showing that anti-CD27 incombination with the TLR3 agonist PolyIC (at 25 μg, 50 μg or 100 μg)enhances T cell responses to an APC-targeted vaccine (α-DEC205-OVA).FIG. 23A is a graph showing the % of IFN-gamma positive cells among CD8+T cells for either wild-type mice treated with poly IC and the anti-CD27mAb 1F5, huCD27-transgenic mice treated with poly IC and a control humanIgG1 antibody or huCD27-transgenic mice treated with poly IC and theanti-CD27 mAb 1F5.

FIGS. 24 and 25 show results from a study of administration of ant-CD27mAb prior to vaccine in the presence or absence of T:LR agnost and showthe significance of timing of the administration of the antibodyrelative to the vaccine.

FIGS. 26 and 27 show results from administration of anti-CD27 mAb incombination with TCR activation ion T-cells from human CD27 transgenicmice, as shown by both proliferation and cytokine production.

FIGS. 28A-D are the protocol for and results of an experiment showingthat anti-CD27 enhances the efficacy of an α-DEC205-OVA vaccine in a MO4(B16-OVA) melanoma challenge model. FIG. 24A shows the protocol for theexperiment. FIG. 24B is a graph plotting the tumor size (in mm²) againstnumber of days post tumor inoculation in untreated mice. FIG. 24C is agraph plotting the tumor size (in mm²) against number of days post tumorinoculation in mice treated with the vaccine alone. FIG. 24D is a graphplotting the tumor size (in mm²) against number of days post tumorinoculation in mice treated with the vaccine in combination with ananti-CD27 antibody.

FIGS. 29A and B are graphs showing prolonged survival of humanCD27-transgenic mice (tumor models) following challenge with a syngeneiclymphoma and administration of various doses of anti-CD27 mAb 1F5.

FIGS. 30A to 32 show the results of an experiment testing the effect ofanti-CD27 treatment in a Raji xenograft model in SCID mice.

FIG. 30A plots the tumor size (in mm³) against number of days post tumorinoculation in mice either untreated, treated with a control human IgG1antibody or treated with the anti-CD27 1F5 and 3H8 antibodies. Arrowsindicate days when antibody treatment was given by i.p. injection.

FIG. 30B shows survival in a Kaplan-Meier plot.

FIG. 31A plots the tumor size (in mm³) against number of days post tumorinoculation in mice either untreated, treated with a control human IgG1antibody or treated with the anti-CD27 1F5 antibody. Arrows indicatedays when antibody treatment was given by i.p. injection. FIG. 31B showssurvival in a Kaplan-Meier plot.

FIG. 32 shows the results of a further experiment testing the effect ofanti-CD27 treatment in a Raji xenograft model in SCID mice in aKaplan-Meier plot.

FIGS. 33A and 33B show the results of an experiment testing the effectof anti-CD27 treatment in a Daudi xenograft model in SCID mice. FIG. 33Aplots the tumor size (in mm³) against number of days post tumorinoculation in mice either treated with a control human IgG1 antibody ortreated with the anti-CD27 1F5 antibody (0.1 mg or 0.3 mg). Arrowsindicate days when antibody treatment was given by i.p. injection. FIG.33B shows survival in a Kaplan-Meier plot.

FIG. 34 shows the results of an ELISPOT assay and that enhancedantigen-specific IFNg production using anti-CD27 antibody is abrogatedwhen the Fc portion of the IgG is unable to engage Fc receptors.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides anti-CD27 antibodies that exhibitparticular functional properties correlating with significanttherapeutic benefits, including upregulation of immune function (e.g. Tcell mediated immune responses as in vaccine therapies, NK activation incancer therapies), inhibition of cell growth (e.g., in cancer therapy)and down-regulation of T cell mediated immune responses (e.g., inautoimmune therapy). These functional features include, for example: (1)inhibition of (e.g., completely or partially blocks) binding of solubleCD70 to CD27 expressing cells by at least about 70%, further for exampleby at least 80% or at least 90% (2) binding to human CD27 with a K_(D)of 1×10⁻⁹ M or less, (3) induction of at least about 40% complementmediated cytotoxicity (CDC) of CD27 expressing cells at a concentrationof 10 μg/ml, (4) induction of at least about 40% specific lysis of CD27expressing cells by ADCC at a concentration of 10 μg/ml, (further forexample at least about 50%, at least about 60% or at least about 70%specific lysis)(5) induction or enhancement of immune responses,especially TH1 responses and/or (6) induction or enhancement of T-cellactivity, especially specific CD8+ T-cell numbers and/or activity. Inother embodiments, the antibodies include particular heavy and lightchain variable regions and/or CDR sequences.

In order that the present invention may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

The term “CD27” (also referred to as “CD27 molecule”, “CD27L receptor”,“S1521”, “T cell activation antigen CD27”, “TNFRSF7,” “MGC20393,” “tumornecrosis factor receptor superfamily, member 7”, “T cell activationantigen 5152” “Tp55”, “Tumor necrosis factor receptor superfamily member7”, “CD27 antigen”, and “T-cell activation antigen CD27”) refers to areceptor that is a member of the TNF-receptor superfamily, which bindsto ligand CD70. CD27 is required for generation and long-termmaintenance of T cell immunity and plays a key role in regulating B-cellactivation and immunoglobulin synthesis. The term “CD27” includes anyvariants or isoforms of CD27 which are naturally expressed by cells(e.g., human CD27 deposited with GENBANK® having accession no.AAH12160.1). Accordingly, antibodies of the invention may cross-reactwith CD27 from species other than human. Alternatively, the antibodiesmay be specific for human CD27 and may not exhibit any cross-reactivitywith other species. CD27 or any variants and isoforms thereof, mayeither be isolated from cells or tissues which naturally express them orbe recombinantly produced using well-known techniques in the art and/orthose described herein. Preferably the antibodies are targeted to hCD27which has a normal glycosylation pattern.

Genbank® (Accession No. AAH12160.1) reports the amino acid sequence ofhuman CD27 as follows (SEQ ID NO:1):

1 marphpwwlc vlgtlvglsa tpapkscper hywaqgklcc qmcepgtflv kdcdqhrkaa 61qcdpcipgvs fspdhhtrph cescrhcnsg llvrnctita naecacrngw qcrdkectec 121dplpnpslta rssqalsphp qpthlpyvse mleartaghm qtladfrqlp artlsthwpp 181qrslcssdfi rilvifsgmf lvftlagalf lhqrrkyrsn kgespvepae pcryscpree 241egstipiqed yrkpepacsp

The term “CD70” (also referred to as “CD70 molecule”, “CD27L”, “CD27LG”,“TNFSF7,” “tumor necrosis factor (ligand) superfamily member 7,” “CD27ligand,” “CD70 antigen,” “surface antigen CD70,” “tumor necrosis factorligand superfamily, member 7,” “Ki-24 antigen,” and “CD27-L”) refers tothe ligand for CD27 (see, for example, Bowman M R et al., J. Immunol.1994 Feb. 15; 152(4):1756-61). CD70 is a type II transmembrane proteinthat belongs to the tumor necrosis factor (TNF) ligand family. It is asurface antigen on activated T and B lymphocytes that inducesproliferation of co-stimulated T cells, enhances the generation ofcytolytic T cells, and contributes to T cell activation. It has alsobeen suggested that CD70 plays a role in regulating B-cell activation,cytotoxic function of natural killer cells, and immunoglobulin synthesis(Hintzen R Q et al., J. Immunol. 1994 Feb. 15; 152(4):1762-73).

Genbank® (Accession No. NP_001243) reports the amino acid sequence ofhuman CD70 as follows (SEQ ID NO: 2):

61 qlnhtgpqqd prlywqggpa lgrsflhgpe ldkgqlrihr dgiymvhiqv tlaicsstta 121srhhpttlav gicspasrsi sllrlsfhqg ctiasqrltp largdtlctn ltgtllpsrn 181tdetffgvqw vrp

The term “antibody” as referred to herein includes whole antibodies andany antigen binding fragment (i.e., “antigen-binding portion”) or singlechain thereof. An “antibody” refers, in one preferred embodiment, to aglycoprotein comprising at least two heavy (H) chains and two light (L)chains inter-connected by disulfide bonds, or an antigen binding portionthereof. Each heavy chain is comprised of a heavy chain variable region(abbreviated herein as V_(H)) and a heavy chain constant region. Theheavy chain constant region is comprised of three domains, CH1, CH2 andCH3. Each light chain is comprised of a light chain variable region(abbreviated herein as V_(L)) and a light chain constant region. Thelight chain constant region is comprised of one domain, CL. The V_(H)and V_(L) regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each V_(H) and V_(L) is composed of three CDRs and fourFRs, arranged from amino-terminus to carboxy-terminus in the followingorder: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of theheavy and light chains contain a binding domain that interacts with anantigen. The constant regions of the antibodies may mediate the bindingof the immunoglobulin to host tissues or factors, including variouscells of the immune system (e.g., effector cells) and the firstcomponent (C1q) of the classical complement system.

The term “antigen-binding portion” of an antibody (or simply “antibodyportion”), as used herein, refers to one or more fragments of anantibody that retain the ability to specifically bind to an antigen(e.g., human CD27). Such “fragments” are, for example between about 8and about 1500 amino acids in length, suitably between about 8 and about745 amino acids in length, suitably about 8 to about 300, for exampleabout 8 to about 200 amino acids, or about 10 to about 50 or 100 aminoacids in length. It has been shown that the antigen-binding function ofan antibody can be performed by fragments of a full-length antibody.Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the V_(L), V_(H), CL and CH1 domains;(ii) a F(ab′)₂ fragment, a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region; (iii) a Fdfragment consisting of the V_(H) and CH1 domains; (iv) a Fv fragmentconsisting of the V_(L) and V_(H) domains of a single arm of anantibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546),which consists of a V_(H) domain; and (vi) an isolated complementaritydetermining region (CDR) or (vii) a combination of two or more isolatedCDRs which may optionally be joined by a synthetic linker. Furthermore,although the two domains of the Fv fragment, V_(L) and V_(H), are codedfor by separate genes, they can be joined, using recombinant methods, bya synthetic linker that enables them to be made as a single proteinchain in which the V_(L) and V_(H) regions pair to form monovalentmolecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988)Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA85:5879-5883). Such single chain antibodies are also intended to beencompassed within the term “antigen-binding portion” of an antibody.These antibody fragments are obtained using conventional techniquesknown to those with skill in the art, and the fragments are screened forutility in the same manner as are intact antibodies. Antigen-bindingportions can be produced by recombinant DNA techniques, or by enzymaticor chemical cleavage of intact immunoglobulins.

A “bispecific” or “bifunctional antibody” is an artificial hybridantibody having two different heavy/light chain pairs and two differentbinding sites. Bispecific antibodies can be produced by a variety ofmethods including fusion of hybridomas or linking of Fab′ fragments.See, e.g., Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321(1990); Kostelny et al., J. Immunol. 148, 1547-1553 (1992).

The term “monoclonal antibody,” as used herein, refers to an antibodywhich displays a single binding specificity and affinity for aparticular epitope. Accordingly, the term “human monoclonal antibody”refers to an antibody which displays a single binding specificity andwhich has variable and optional constant regions derived from humangermline immunoglobulin sequences. In one embodiment, human monoclonalantibodies are produced by a hybridoma which includes a B cell obtainedfrom a transgenic non-human animal, e.g., a transgenic mouse, having agenome comprising a human heavy chain transgene and a light chaintransgene fused to an immortalized cell.

The term “recombinant human antibody,” as used herein, includes allhuman antibodies that are prepared, expressed, created or isolated byrecombinant means, such as (a) antibodies isolated from an animal (e.g.,a mouse) that is transgenic or transchromosomal for human immunoglobulingenes or a hybridoma prepared therefrom, (b) antibodies isolated from ahost cell transformed to express the antibody, e.g., from atransfectoma, (c) antibodies isolated from a recombinant, combinatorialhuman antibody library, and (d) antibodies prepared, expressed, createdor isolated by any other means that involve splicing of humanimmunoglobulin gene sequences to other DNA sequences. Such recombinanthuman antibodies comprise variable and constant regions that utilizeparticular human germline immunoglobulin sequences are encoded by thegermline genes, but include subsequent rearrangements and mutationswhich occur, for example, during antibody maturation. As known in theart (see, e.g., Lonberg (2005) Nature Biotech. 23(9):1117-1125), thevariable region contains the antigen binding domain, which is encoded byvarious genes that rearrange to form an antibody specific for a foreignantigen. In addition to rearrangement, the variable region can befurther modified by multiple single amino acid changes (referred to assomatic mutation or hypermutation) to increase the affinity of theantibody to the foreign antigen. The constant region will change infurther response to an antigen (i.e., isotype switch). Therefore, therearranged and somatically mutated nucleic acid molecules that encodethe light chain and heavy chain immunoglobulin polypeptides in responseto an antigen may not have sequence identity with the original nucleicacid molecules, but instead will be substantially identical or similar(i.e., have at least 80% identity).

The term “human antibody” includes antibodies having variable andconstant regions (if present) of human germline immunoglobulinsequences. Human antibodies of the invention can include amino acidresidues not encoded by human germline immunoglobulin sequences (e.g.,mutations introduced by random or site-specific mutagenesis in vitro orby somatic mutation in vivo) (see, Lonberg, N. et al. (1994) Nature368(6474): 856-859); Lonberg, N. (1994) Handbook of ExperimentalPharmacology 113:49-101; Lonberg, N. and Huszar, D. (1995) Intern. Rev.Immunol. Vol. 13: 65-93, and Harding, F. and Lonberg, N. (1995) Ann.N.Y. Acad. Sci 764:536-546). However, the term “human antibody” does notinclude antibodies in which CDR sequences derived from the germline ofanother mammalian species, such as a mouse, have been grafted onto humanframework sequences (i.e., humanized antibodies).

As used herein, a “heterologous antibody” is defined in relation to thetransgenic non-human organism producing such an antibody. This termrefers to an antibody having an amino acid sequence or an encodingnucleic acid sequence corresponding to that found in an organism notconsisting of the transgenic non-human animal, and generally from aspecies other than that of the transgenic non-human animal.

An “isolated antibody,” as used herein, is intended to refer to anantibody which is substantially free of other antibodies havingdifferent antigenic specificities (e.g., an isolated antibody thatspecifically binds to human CD27 is substantially free of antibodiesthat specifically bind antigens other than human CD27). An isolatedantibody that specifically binds to an epitope of may, however, havecross-reactivity to other CD27 proteins from different species. However,the antibody preferably always binds to human CD27. In addition, anisolated antibody is typically substantially free of other cellularmaterial and/or chemicals. In one embodiment of the invention, acombination of “isolated” antibodies having different CD27 specificitiesis combined in a well defined composition.

The term “epitope” or “antigenic determinant” refers to a site on anantigen to which an immunoglobulin or antibody specifically binds.Epitopes can be formed both from contiguous amino acids or noncontiguousamino acids juxtaposed by tertiary folding of a protein. Epitopes formedfrom contiguous amino acids are typically retained on exposure todenaturing solvents, whereas epitopes formed by tertiary folding aretypically lost on treatment with denaturing solvents. An epitopetypically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or15 amino acids in a unique spatial conformation. Methods for determiningwhat epitopes are bound by a given antibody (i.e., epitope mapping) arewell known in the art and include, for example, immunoblotting andimmunoprecipitation assays, wherein overlapping or contiguous peptidesfrom CD27 are tested for reactivity with the given anti-CD27 antibody.Methods of determining spatial conformation of epitopes includetechniques in the art and those described herein, for example, x-raycrystallography and 2-dimensional nuclear magnetic resonance (see, e.g.,Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996)).

Also, encompassed by the present invention are antibodies that bind toan epitope on CD27 which comprises all or a portion of an epitoperecognized by the particular antibodies described herein (e.g., the sameor an overlapping region or a region between or spanning the region).

Also encompassed by the present invention are antibodies that bind thesame epitope and/or antibodies that compete for binding to human CD27with the antibodies described herein. Antibodies that recognize the sameepitope or compete for binding can be identified using routinetechniques. Such techniques include, for example, an immunoassay, whichshows the ability of one antibody to block the binding of anotherantibody to a target antigen, i.e., a competitive binding assay.Competitive binding is determined in an assay in which theimmunoglobulin under test inhibits specific binding of a referenceantibody to a common antigen, such as CD27. Numerous types ofcompetitive binding assays are known, for example: solid phase direct orindirect radioimmunoassay (RIA), solid phase direct or indirect enzymeimmunoassay (EIA), sandwich competition assay (see Stahli et al.,Methods in Enzymology 9:242 (1983)); solid phase direct biotin-avidinEIA (see Kirkland et al., J. Immunol. 137:3614 (1986)); solid phasedirect labeled assay, solid phase direct labeled sandwich assay (seeHarlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborPress (1988)); solid phase direct label RIA using 1-125 label (see Morelet al., Mol. Immunol. 25(1):7 (1988)); solid phase direct biotin-avidinEIA (Cheung et al., Virology 176:546 (1990)); and direct labeled RIA.(Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)). Typically, suchan assay involves the use of purified antigen bound to a solid surfaceor cells bearing either of these, an unlabeled test immunoglobulin and alabeled reference immunoglobulin. Competitive inhibition is measured bydetermining the amount of label bound to the solid surface or cells inthe presence of the test immunoglobulin. Usually the test immunoglobulinis present in excess. Usually, when a competing antibody is present inexcess, it will inhibit specific binding of a reference antibody to acommon antigen by at least 50-55%, 55-60%, 60-65%, 65-70% 70-75% ormore.

Other techniques include, for example, epitope mapping methods, such as,x-ray analyses of crystals of antigen:antibody complexes which providesatomic resolution of the epitope. Other methods monitor the binding ofthe antibody to antigen fragments or mutated variations of the antigenwhere loss of binding due to a modification of an amino acid residuewithin the antigen sequence is often considered an indication of anepitope component. In addition, computational combinatorial methods forepitope mapping can also be used. These methods rely on the ability ofthe antibody of interest to affinity isolate specific short peptidesfrom combinatorial phage display peptide libraries. The peptides arethen regarded as leads for the definition of the epitope correspondingto the antibody used to screen the peptide library. For epitope mapping,computational algorithms have also been developed which have been shownto map conformational discontinuous epitopes.

As used herein, the terms “specific binding,” “selective binding,”“selectively binds,” and “specifically binds,” refer to antibody bindingto an epitope on a predetermined antigen. Typically, the antibody bindswith an equilibrium dissociation constant (K_(D)) of approximately lessthan 10⁻⁷ M, such as approximately less than 10⁻⁸ M, 10⁻⁹ M or 10⁻¹⁰ Mor even lower when determined by surface plasmon resonance (SPR)technology in a BIACORE 2000 instrument using recombinant human CD27 asthe analyte and the antibody as the ligand and binds to thepredetermined antigen with an affinity that is at least two-fold greaterthan its affinity for binding to a non-specific antigen (e.g., BSA,casein) other than the predetermined antigen or a closely-relatedantigen. The phrases “an antibody recognizing an antigen” and “anantibody specific for an antigen” are used interchangeably herein withthe term “an antibody which binds specifically to an antigen.”

The term “K_(D),” as used herein, is intended to refer to thedissociation equilibrium constant of a particular antibody-antigeninteraction. Typically, the human antibodies of the invention bind toCD27 with a dissociation equilibrium constant (K_(D)) of approximately10⁻⁸ M or less, such as less than 10⁻⁹ M or 10⁻¹⁰ M or even lower whendetermined by surface plasmon resonance (SPR) technology in a BIACORE2000 instrument using recombinant human CD27 as the analyte and theantibody as the ligand.

The term “kd” as used herein, is intended to refer to the off rateconstant for the dissociation of an antibody from the antibody/antigencomplex.

The term “ka” as used herein, is intended to refer to the on rateconstant for the association of an antibody with the antigen.

The term “EC50,” as used herein, refers to the concentration of anantibody or an antigen-binding portion thereof, which induces aresponse, either in an in vitro or an in vivo assay, which is 50% of themaximal response, i.e., halfway between the maximal response and thebaseline.

As used herein, “isotype” refers to the antibody class (e.g., IgM orIgG1) that is encoded by heavy chain constant region genes. In oneembodiment, a human monoclonal antibody of the invention is of the IgG1isotype. In another embodiment, a human monoclonal antibody of theinvention is of the IgG2 isotype.

The term “binds to immobilized CD27,” refers to the ability of a humanantibody of the invention to bind to CD27, for example, expressed on thesurface of a cell or which is attached to a solid support.

The term “cross-reacts,” as used herein, refers to the ability of anantibody of the invention to bind to CD27 from a different species. Forexample, an antibody of the present invention which binds human CD27 mayalso bind another species of CD27. As used herein, cross-reactivity ismeasured by detecting a specific reactivity with purified antigen inbinding assays (e.g., SPR, ELISA) or binding to, or otherwisefunctionally interacting with, cells physiologically expressing CD27.Methods for determining cross-reactivity include standard binding assaysas described herein, for example, by Biacore™ surface plasmon resonance(SPR) analysis using a Biacore™ 2000 SPR instrument (Biacore AB,Uppsala, Sweden), or flow cytometric techniques.

As used herein, “isotype switching” refers to the phenomenon by whichthe class, or isotype, of an antibody changes from one Ig class to oneof the other Ig classes.

As used herein, “nonswitched isotype” refers to the isotypic class ofheavy chain that is produced when no isotype switching has taken place;the CH gene encoding the nonswitched isotype is typically the first CHgene immediately downstream from the functionally rearranged VDJ gene.Isotype switching has been classified as classical or non-classicalisotype switching. Classical isotype switching occurs by recombinationevents which involve at least one switch sequence region in thetransgene. Non-classical isotype switching may occur by, for example,homologous recombination between human σ_(μ) and human Σ_(μ)(δ-associated deletion). Alternative non-classical switching mechanisms,such as intertransgene and/or interchromosomal recombination, amongothers, may occur and effectuate isotype switching.

As used herein, the term “switch sequence” refers to those DNA sequencesresponsible for switch recombination. A “switch donor” sequence,typically a μ switch region, will be 5′ (i.e., upstream) of theconstruct region to be deleted during the switch recombination. The“switch acceptor” region will be between the construct region to bedeleted and the replacement constant region (e.g., γ, ε, etc.). As thereis no specific site where recombination always occurs, the final genesequence will typically not be predictable from the construct.

As used herein, “glycosylation pattern” is defined as the pattern ofcarbohydrate units that are covalently attached to a protein, morespecifically to an immunoglobulin protein. A glycosylation pattern of aheterologous antibody can be characterized as being substantiallysimilar to glycosylation patterns which occur naturally on antibodiesproduced by the species of the nonhuman transgenic animal, when one ofordinary skill in the art would recognize the glycosylation pattern ofthe heterologous antibody as being more similar to said pattern ofglycosylation in the species of the nonhuman transgenic animal than tothe species from which the CH genes of the transgene were derived.

The term “naturally-occurring” as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory isnaturally-occurring.

The term “rearranged” as used herein refers to a configuration of aheavy chain or light chain immunoglobulin locus wherein a V segment ispositioned immediately adjacent to a D-J or J segment in a conformationencoding essentially a complete V_(H) or V_(L) domain, respectively. Arearranged immunoglobulin gene locus can be identified by comparison togermline DNA; a rearranged locus will have at least one recombinedheptamer/nonamer homology element.

The term “unrearranged” or “germline configuration” as used herein inreference to a V segment refers to the configuration wherein the Vsegment is not recombined so as to be immediately adjacent to a D or Jsegment.

The term “nucleic acid molecule,” as used herein, is intended to includeDNA molecules and RNA molecules. A nucleic acid molecule may besingle-stranded or double-stranded, but preferably is double-strandedDNA.

The term “isolated nucleic acid molecule,” as used herein in referenceto nucleic acids encoding antibodies or antibody portions (e.g., V_(H),V_(L), CDR3) that bind to CD27, is intended to refer to a nucleic acidmolecule in which the nucleotide sequences encoding the antibody orantibody portion are free of other nucleotide sequences encodingantibodies or antibody portions that bind antigens other than CD27,which other sequences may naturally flank the nucleic acid in humangenomic DNA. For example, SEQ ID NOs: 35 and 41, 47 and 53, 101 and 107,83 and 89, 83 and 95, 23 and 29, 71 and 77 correspond, respectively, tothe nucleotide sequences comprising the heavy chain (V_(H)) and lightchain (VL) variable regions of anti-CD27 antibody monoclonal antibodies1F5, 1H8, 3H12, 3A10, 2C2, 2G9, 3H8, and 1G5.

The present invention also encompasses “conservative sequencemodifications” of the sequences set forth in SEQ ID NOs: 5-112, i.e.,nucleotide and amino acid sequence modifications which do not abrogatethe binding of the antibody encoded by the nucleotide sequence orcontaining the amino acid sequence, to the antigen. Such conservativesequence modifications include conservative nucleotide and amino acidsubstitutions, as well as, nucleotide and amino acid additions anddeletions. For example, modifications can be introduced into SEQ ID NOs:5-112 by standard techniques known in the art, such as site-directedmutagenesis and PCR-mediated mutagenesis. Conservative amino acidsubstitutions include ones in which the amino acid residue is replacedwith an amino acid residue having a similar side chain. Families ofamino acid residues having similar side chains have been defined in theart. These families include amino acids with basic side chains (e.g.,lysine, arginine, histidine), acidic side chains (e.g., aspartic acid,glutamic acid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolarside chains (e.g., alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Thus, a predicted nonessentialamino acid residue in a human anti-CD27 antibody is preferably replacedwith another amino acid residue from the same side chain family. Methodsof identifying nucleotide and amino acid conservative substitutionswhich do not eliminate antigen binding are well-known in the art (see,e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al.Protein Eng. 12(10):879-884 (1999); and Burks et al. Proc. Natl. Acad.Sci. USA 94:412-417 (1997))

Alternatively, in another embodiment, mutations can be introducedrandomly along all or part of an anti-CD27 antibody coding sequence,such as by saturation mutagenesis, and the resulting modified anti-CD27antibodies can be screened for binding activity.

For nucleic acids, the term “substantial homology” indicates that twonucleic acids, or designated sequences thereof, when optimally alignedand compared, are identical, with appropriate nucleotide insertions ordeletions, in at least about 80% of the nucleotides, usually at leastabout 90% to 95%, and more preferably at least about 98% to 99.5% of thenucleotides. Alternatively, substantial homology exists when thesegments will hybridize under selective hybridization conditions, to thecomplement of the strand.

The percent identity between two sequences is a function of the numberof identical positions shared by the sequences (i.e., % homology=# ofidentical positions/total # of positions×100), taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences. The comparison of sequencesand determination of percent identity between two sequences can beaccomplished using a mathematical algorithm, as described in thenon-limiting examples below.

The percent identity between two nucleotide sequences can be determinedusing the GAP program in the GCG software package (available athttp://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. Thepercent identity between two nucleotide or amino acid sequences can alsobe determined using the algorithm of E. Meyers and W. Miller (CABIOS,4:11-17 (1989)) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4. In addition, the percent identity betweentwo amino acid sequences can be determined using the Needleman andWunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat http://www.gcg.com), using either a Blossum 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6.

The nucleic acid and protein sequences of the present invention canfurther be used as a “query sequence” to perform a search against publicdatabases to, for example, identify related sequences. Such searches canbe performed using the NBLAST and XBLAST programs (version 2.0) ofAltschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotidesearches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous to the nucleicacid molecules of the invention. BLAST protein searches can be performedwith the XBLAST program, score=50, wordlength=3 to obtain amino acidsequences homologous to the protein molecules of the invention. Toobtain gapped alignments for comparison purposes, Gapped BLAST can beutilized as described in Altschul et al., (1997) Nucleic Acids Res.25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, thedefault parameters of the respective programs (e.g., XBLAST and NBLAST)can be used. See http://www.ncbi.nlm.nih.gov.

The nucleic acids may be present in whole cells, in a cell lysate, or ina partially purified or substantially pure form. A nucleic acid is“isolated” or “rendered substantially pure” when purified away fromother cellular components or other contaminants, e.g., other cellularnucleic acids or proteins, by standard techniques, includingalkaline/SDS treatment, CsCl banding, column chromatography, agarose gelelectrophoresis and others well known in the art. See, F. Ausubel, etal., ed. Current Protocols in Molecular Biology, Greene Publishing andWiley Interscience, New York (1987).

The nucleic acid compositions of the present invention, while often in anative sequence (except for modified restriction sites and the like),from either cDNA, genomic or mixtures thereof may be mutated, inaccordance with standard techniques to provide gene sequences. Forcoding sequences, these mutations, may affect amino acid sequence asdesired. In particular, DNA sequences substantially homologous to orderived from native V, D, J, constant, switches and other such sequencesdescribed herein are contemplated (where “derived” indicates that asequence is identical or modified from another sequence).

A nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For instance, apromoter or enhancer is operably linked to a coding sequence if itaffects the transcription of the sequence. With respect to transcriptionregulatory sequences, operably linked means that the DNA sequences beinglinked are contiguous and, where necessary to join two protein codingregions, contiguous and in reading frame. For switch sequences, operablylinked indicates that the sequences are capable of effecting switchrecombination.

The term “vector,” as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid,” which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”) In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” may be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

The term “recombinant host cell” (or simply “host cell”), as usedherein, is intended to refer to a cell into which a recombinantexpression vector has been introduced. It should be understood that suchterms are intended to refer not only to the particular subject cell butto the progeny of such a cell. Because certain modifications may occurin succeeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term “host cell” asused herein.

As used herein, the term “antigen” refers to any natural or syntheticimmunogenic substance, such as a protein, peptide, or hapten. Suitableantigens for use in the present invention (e.g., in a vaccine incombination with an anti-CD27 antibody of the invention) include, forexample, infectious disease antigens and tumor antigens, against whichprotective or therapeutic immune responses are desired, e.g., antigensexpressed by a tumor cell or a pathogenic organism or infectious diseaseantigens. For example, suitable antigens include tumor-associatedantigens for the prevention or treatment of cancers. Examples oftumor-associated antigens include, but are not limited to, sequencescomprising all or part of the sequences of βhCG, gp100 or Pmel17,HER2/neu, WT1, mesothelin, CEA, gp100, MART1, TRP-2, melan-A, NY-ESO-1,NY-BR-1, NY-CO-58, MN (gp250), idiotype, MAGE-1, MAGE-3, MAGE-A3,Tyrosinase, Telomerase, SSX2 and MUC-1 antigens, and germ cell derivedtumor antigens. Tumor associated antigens also include the blood groupantigens, for example, Le^(a), Le^(b), LeX, LeY, H-2, B-1, B-2 antigens.Alternatively, more than one antigen can be included within theantigen-antibody constructs of the invention. For example, a MAGEantigen can be combined with other antigens such as melanin A,tyrosinase, and gp100 along with adjuvants such as GM-CSF or IL-12, andlinked to an anti-APC antibody.

Other suitable antigens include viral antigens for the prevention ortreatment of viral diseases. Examples of viral antigens include, but arenot limited to, HIV-1 gag, HIV-1 env, HIV-1 nef, HBV (surface or coreantigens), HPV, FAS, HSV-1, HSV-2, p17, ORF2 and ORF3 antigens. Examplesof bacterial antigens include, but are not limited to, Toxoplasma gondiior Treponema pallidum. The antibody-bacterial antigen conjugates of theinvention can be in the treatment or prevention of various bacterialdiseases such as Anthrax, Botulism, Tetanus, Chlamydia, Cholera,Diphtheria, Lyme Disease, Syphilis and Tuberculosis. Other suitableantigens from infectious disease pathogens, such as viruses, bacteria,parasites and fungi are disclosed below.

Sequences of the foregoing antigens are well known in the art. Forexample, an example of a MAGE-3 cDNA sequence is provided in U.S. Pat.No. 6,235,525 (Ludwig Institute for Cancer Research); examples ofNY-ESO-1 nucleic acid and protein sequences are provided in U.S. Pat.Nos. 5,804,381 and 6,069,233 (Ludwig Institute for Cancer Research);examples of Melan-A nucleic acid and protein sequences are provided inU.S. Pat. Nos. 5,620,886 and 5,854,203 (Ludwig Institute for CancerResearch); examples of NY-BR-1 nucleic acid and protein sequences areprovided in U.S. Pat. Nos. 6,774,226 and 6,911,529 (Ludwig Institute forCancer Research) and examples of NY-CO-58 nucleic acid and proteinsequences are provided in WO 02090986 (Ludwig Institute for CancerResearch); an example of an amino acid sequence for the HER-2/neuprotein is available at GENBANK® Accession No. AAA58637; and anucleotide sequence (mRNA) for human carcinoembryonic antigen-like 1(CEA-1) is available at GENBANK® Accession No. NM_020219.

An HPV antigen that may be used in the compositions and the methods ofthe invention may include, for example an HPV-16 antigen, an HPV-18antigen, an HPV-31 antigen, an HPV-33 antigen and/or an HPV-35 antigen;and is suitably an HPV-16 antigen and/or HPV-18 antigen. A genome ofHPV-16 is described in Virology, 145:181-185 (1985) and DNA sequencesencoding HPV-18 are described in U.S. Pat. No. 5,840,306, thedisclosures of which are incorporated by reference herein in theirentirety. HPV-16 antigens (e.g., seroreactive regions of the E1 and/orE2 proteins of HPV-16) are described in U.S. Pat. No. 6,531,127, andHPV-18 antigens (e.g., seroreactive regions of the L1 and/or L2 proteinsof HPV-18) are described in U.S. Pat. No. 5,840,306, the disclosures ofwhich are incorporated by reference herein. Similarly, a complete genomefor HBV is available at GENBANK® Accession No. NC 003977, the disclosureof which is incorporated herein. The genome of HCV is described inEuropean Patent Application No. 318 216, the disclosure of which isincorporated herein. PCT/US90/01348, incorporated by reference herein,discloses sequence information of clones of the HCV genome, amino acidsequences of HCV viral proteins and methods of making and using suchcompositions for HCV vaccines comprising HCV proteins and peptidesderived there from.

Antigenic peptides of proteins (i.e., those containing T cell epitopes)can be identified in a variety of manners well known in the art. Forexample, T cell epitopes can be predicted by analyzing the sequence ofthe protein using web-based predictive algorithms (BIMAS & SYFPEITHI) togenerate potential MHC class I and II-binding peptides that match aninternal database of 10,000 well characterized MHC binding peptidespreviously defined by CTLs. High scoring peptides can be ranked andselected as “interesting” on the basis of high affinity to a given MHCmolecule.

Another method for identifying antigenic peptides containing T cellepitopes is by dividing the protein into non-overlapping peptides ofdesired length or overlapping peptides of desired lengths which can beproduced recombinantly, synthetically, or in certain limited situations,by chemical cleavage of the protein and tested for immunogenicproperties, e.g., eliciting a T cell response (i.e., proliferation orlymphokine secretion).

In order to determine precise T cell epitopes of the protein by, forexample, fine mapping techniques, a peptide having T cell stimulatingactivity and thus comprising at least one T cell epitope, as determinedby T cell biology techniques, can be modified by addition or deletion ofamino acid residues at either the amino or carboxy terminus of thepeptide and tested to determine a change in T cell reactivity to themodified peptide. If two or more peptides which share an area of overlapin the native protein sequence are found to have human T cellstimulating activity, as determined by T cell biology techniques,additional peptides can be produced comprising all or a portion of suchpeptides and these additional peptides can be tested by a similarprocedure. Following this technique, peptides are selected and producedrecombinantly or synthetically. Peptides are selected based on variousfactors, including the strength of the T cell response to the peptide(e.g., stimulation index). The physical and chemical properties of theseselected peptides (e.g., solubility, stability) can then be examined todetermine whether the peptides are suitable for use in therapeuticcompositions or whether the peptides require modification.

The term “antigen presenting cell” or “APC” is a cell that displaysforeign antigen complexed with MHC on its surface. T-cells recognizethis complex using T-cell receptor (TCR). Examples of APCs include, butare not limited to, dendritic cells (DCs), peripheral blood mononuclearcells (PBMC), monocytes (such as THP-1), B lymphoblastoid cells (such asC1R.A2, 1518 B-LCL) and monocyte-derived dendritic cells (DCs). SomeAPCs internalize antigens either by phagocytosis or by receptor-mediatedendocytosis. Examples of APC receptors include, but are not limited toC-type lectins, such as, the human Dendritic and Epithelial Cell 205receptor (CD27), and the human macrophage mannose receptor.

The term “antigen presentation” refers to the process by which APCscapture antigens and enables their recognition by T-cells, e.g., as acomponent of an MHC-I and/or MHC-II conjugate.

“MHC molecules” include two types of molecules, MHC class I and MHCclass II. MHC class I molecules present antigen to specific CD8+ T cellsand MHC class II molecules present antigen to specific CD4+ T cells.Antigens delivered exogenously to APCs are processed primarily forassociation with MHC class II. In contrast, antigens deliveredendogenously to APCs are processed primarily for association with MHCclass I.

As used herein, the term “immunostimulatory agent” includes but is notlimited to compounds capable of stimulating APCs, such as DCs andmacrophages. For example, suitable immunostimulatory agents for use inthe present invention are capable of stimulating APCs, so that thematuration process of the APCs is accelerated, the proliferation of APCsis increased, and/or the recruitment or release of co-stimulatorymolecules (e.g., CD80, CD86, ICAM-1, MHC molecules and CCR7) andpro-inflammatory cytokines (e.g., IL-1β, IL-6, IL-12, IL-15, and IFN-γ)is upregulated. Suitable immunostimulatory agents are also capable ofincreasing T cell proliferation. Such immunostimulatory agents include,but are not be limited to, CD40 ligand; FLT 3 ligand; cytokines, such asIFN-α, IFN-β, IFN-γ and IL-2; colony-stimulating factors, such as G-CSF(granulocyte colony-stimulating factor) and GM-CSF(granulocyte-macrophage colony-stimulating factor); an anti-CTLA-4antibody, anti-PD1 antibody, anti-41BB antibody, or anti-OX-40 antibody;LPS (endotoxin); ssRNA; dsRNA; Bacille Calmette-Guerin (BCG); Levamisolehydrochloride; and intravenous immune globulins. In one embodiment animmunostimulatory agant may be a Toll-like Receptor (TLR) agonist. Forexample the immunostimulatory agent may be a TLR3 agonist such asdouble-stranded inosine:cytosine polynucleotide (Poly I:C, for exampleavailable as Ampligen™ from Hemispherx Bipharma, PA, US or Poly IC:LCfrom Oncovir) or Poly A:U; a TLR4 agonist such as monophosphoryl lipid A(MPL) or RC-529 (for example as available from GSK, UK); a TLR5 agonistsuch as flagellin; a TLR7 or TLR8 agonist such as an imidazoquinolineTLR7 or TLR 8 agonist, for example imiquimod (eg Aldara™) or resiquimodand related imidazoquinoline agents (for example as available from 3MCorporation); or a TLR 9 agonist such as a deoxynucleotide withunmethylated CpG motifs (so-called “CpGs”, for example as available fromColey Pharmaceutical). A preferred immunostimulatory agent is a TLR3agonist, preferably Poly I:C. Such immunostimulatory agents may beadministered simultaneously, separately or sequentially with theantibodies and constructs of the present invention and may also bephysically linked to the antibodies and constructs.

As used herein, the term “linked” refers to the association of two ormore molecules. The linkage can be covalent or non-covalent. The linkagealso can be genetic (i.e., recombinantly fused). Such linkages can beachieved using a wide variety of art recognized techniques, such aschemical conjugation and recombinant protein production.

As used herein, the term antigen “cross-presentation” refers topresentation of exogenous protein antigens to T cells via MHC class Iand class II molecules on APCs.

As used herein, the term “T cell-mediated response” refers to anyresponse mediated by T cells, including effector T cells (e.g., CD8⁺cells) and helper T cells (e.g., CD4⁺ cells). T cell mediated responsesinclude, for example, T cell cytotoxicity and proliferation.

As used herein, the term “cytotoxic T lymphocyte (CTL) response” refersto an immune response induced by cytotoxic T cells. CTL responses aremediated primarily by CD8⁺ T cells.

As used herein, the terms “inhibits” or “blocks” (e.g., referring toinhibition/blocking of binding of CD70 to CD27 on cells) are usedinterchangeably and encompass both partial and completeinhibition/blocking. The inhibition/blocking of CD70 preferably reducesor alters the normal level or type of activity that occurs when CD70binding occurs without inhibition or blocking. Inhibition and blockingare also intended to include any measurable decrease in the bindingaffinity of CD70 when in contact with an anti-CD27 antibody as comparedto CD70 not in contact with an anti-CD27 antibody, e.g., inhibitsbinding of CD70 by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,99%, or 100%. In a preferred embodiment, the anti-CD27 antibody inhibitsbinding of CD70 by at least about 70%. In another embodiment, theanti-CD27 antibody inhibits binding of CD70 by at least 80%

As used herein, the term “inhibits growth” (e.g., referring to cells) isintended to include any measurable decrease in the growth of a cell,e.g., the inhibition of growth of a cell by at least about 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%.

The terms “inducing an immune response” and “enhancing an immuneresponse” are used interchangeably and refer the stimulation of animmune response (i.e., either passive or adaptive) to a particularantigen. The terms “induce” as used with respect to inducing CDC or ADCCrefer to the stimulation of particular direct cell killing mechanisms.For example, in one embodiment, the antibody induces at least about 20,25, 30, 35, 40, 45, 50, 55, or 60% lysis via CDC of CD27 expressingcells at a concentration of 10 μg/ml. In a preferred embodiment, theantibody induces at least about 40% lysis via CDC of CD27 expressingcells at a concentration of 10 μg/ml. In another embodiment, theantibody induces at least about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, or 85% lysis via ADCC (i.e., specific lysis) of CD27expressing cells at a concentration of 10 μg/ml. In a preferredembodiment, the antibody induces at least about 40% lysis via ADCC ofCD27 expressing cells at a concentration of 10 μg/ml.

The terms “treat,” “treating,” and “treatment,” as used herein, refer totherapeutic or preventative measures described herein. The methods of“treatment” employ administration to a subject, in need of suchtreatment, a human antibody of the present invention, for example, asubject in need of an enhanced immune response against a particularantigen or a subject who ultimately may acquire such a disorder, inorder to prevent, cure, delay, reduce the severity of, or ameliorate oneor more symptoms of the disorder or recurring disorder, or in order toprolong the survival of a subject beyond that expected in the absence ofsuch treatment.

The term “effective dose” or “effective dosage” is defined as an amountsufficient to achieve or at least partially achieve the desired effect.The term “therapeutically effective dose” is defined as an amountsufficient to cure or at least partially arrest the disease and itscomplications in a patient already suffering from the disease. Amountseffective for this use will depend upon the severity of the disorderbeing treated and the general state of the patient's own immune system.

The term “patient” includes human and other mammalian subjects thatreceive either prophylactic or therapeutic treatment.

As used herein, the term “subject” includes any human or non-humananimal. For example, the methods and compositions of the presentinvention can be used to treat a subject with an immune disorder. Theterm “non-human animal” includes all vertebrates, e.g., mammals andnon-mammals, such as non-human primates, sheep, dog, cow, chickens,amphibians, reptiles, etc.

Various aspects of the invention are described in further detail in thefollowing subsections.

I. Production of Antibodies to CD27

The present invention encompasses antibodies, e.g., fully humanantibodies, that bind CD27, e.g., human CD27. Exemplary monoclonalantibodies that bind CD27 include 1F5, 1H8, 3H12, 3A10, 2C2, 2G9, 3H8,and 1G5. Monoclonal antibodies of the invention can be produced using avariety of known techniques, such as the standard somatic cellhybridization technique described by Kohler and Milstein, Nature 256:495 (1975). Although somatic cell hybridization procedures arepreferred, in principle, other techniques for producing monoclonalantibodies also can be employed, e.g., viral or oncogenic transformationof B lymphocytes, phage display technique using libraries of humanantibody genes.

Accordingly, in one embodiment, a hybridoma method is used for producingan antibody that binds human CD27. In this method, a mouse or otherappropriate host animal can be immunized with a suitable antigen inorder to elicit lymphocytes that produce or are capable of producingantibodies that will specifically bind to the antigen used forimmunization. Alternatively, lymphocytes may be immunized in vitro.Lymphocytes can then be fused with myeloma cells using a suitable fusingagent, such as polyethylene glycol, to form a hybridoma cell (Goding,Monoclonal Antibodies: Principles and Practice, pp. 59-103 (AcademicPress, 1986)). Culture medium in which hybridoma cells are growing isassayed for production of monoclonal antibodies directed against theantigen. After hybridoma cells are identified that produce antibodies ofthe desired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103(Academic Press, 1986)). Suitable culture media for this purposeinclude, for example, D-MEM or RPMI-1640 medium. In addition, thehybridoma cells may be grown in vivo as ascites tumors in an animal. Themonoclonal antibodies secreted by the subclones can be separated fromthe culture medium, ascites fluid, or serum by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

In another embodiment, antibodies and antibody portions that bind humanCD27 can be isolated from antibody phage libraries generated using thetechniques described in, for example, McCafferty et al., Nature,348:552-554 (1990). Clackson et al., Nature, 352:624-628 (1991), Markset al., J. Mol. Biol., 222:581-597 (1991) and Hoet et al (2005) NatureBiotechnology 23, 344-348; U.S. Pat. Nos. 5,223,409; 5,403,484; and5,571,698 to Ladner et al.; U.S. Pat. Nos. 5,427,908 and 5,580,717 toDower et al.; U.S. Pat. Nos. 5,969,108 and 6,172,197 to McCafferty etal.; and U.S. Pat. Nos. 5,885,793; 6,521,404; 6,544,731; 6,555,313;6,582,915 and 6,593,081 to Griffiths et al. Additionally, production ofhigh affinity (nM range) human antibodies by chain shuffling (Marks etal., Bio/Technology, 10:779-783 (1992)), as well as combinatorialinfection and in vivo recombination as a strategy for constructing verylarge phage libraries (Waterhouse et al., Nuc. Acids. Res., 21:2265-2266(1993)) may also be used.

In a particular embodiment, the antibody that binds human CD27 isproduced using the phage display technique described by Hoet et al.,supra. This technique involves the generation of a human Fab libraryhaving a unique combination of immunoglobulin sequences isolated fromhuman donors and having synthetic diversity in the heavy-chain CDRs isgenerated. The library is then screened for Fabs that bind to humanCD27.

The preferred animal system for generating hybridomas which produceantibodies of the invention is the murine system. Hybridoma productionin the mouse is well known in the art, including immunization protocolsand techniques for isolating and fusing immunized splenocytes.

In one embodiment, antibodies directed against CD27 are generated usingtransgenic or transchromosomal mice carrying parts of the human immunesystem rather than the mouse system. In one embodiment, the inventionemploys transgenic mice, referred to herein as “HuMAb mice” whichcontain a human immunoglobulin gene miniloci that encodes unrearrangedhuman heavy (μ and γ) and κ light chain immunoglobulin sequences,together with targeted mutations that inactivate the endogenous μ and κchain loci (Lonberg, N. et al. (1994) Nature 368(6474): 856-859).Accordingly, the mice exhibit reduced expression of mouse IgM or κ, andin response to immunization, the introduced human heavy and light chaintransgenes undergo class switching and somatic mutation to generate highaffinity human IgGK monoclonal antibodies (Lonberg, N. et al. (1994),supra; reviewed in Lonberg, N. (1994) Handbook of ExperimentalPharmacology 113:49-101; Lonberg, N. and Huszar, D. (1995) Intern. Rev.Immunol. Vol. 13: 65-93, and Harding, F. and Lonberg, N. (1995) Ann.N.Y. Acad. Sci 764:536-546). The preparation of HuMAb mice is describedin detail in Section II below and in Taylor, L. et al. (1992) NucleicAcids Research 20:6287-6295; Chen, J. et al. (1993) InternationalImmunology 5: 647-656; Tuaillon et al. (1993) Proc. Natl. Acad. Sci USA90:3720-3724; Choi et al. (1993) Nature Genetics 4:117-123; Chen, J. etal. (1993) EMBO J. 12: 821-830; Tuaillon et cd. (1994) J. Immunol.152:2912-2920; Lonberg et al., (1994) Nature 368(6474): 856-859;Lonberg, N. (1994) Handbook of Experimental Pharmacology 113:49-101;Taylor, L. et al. (1994) International Immunology 6: 579-591; Lonberg,N. and Huszar, D. (1995) Intern. Rev. Immunol. Vol. 13: 65-93; Harding,F. and Lonberg, N. (1995) Ann. N.Y. Acad. Sci 764:536-546; Fishwild, D.et al. (1996) Nature Biotechnology 14: 845-851. See further, U.S. Pat.Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397;5,661,016; 5,814,318; 5,874,299; and 5,770,429; all to Lonberg and Kay,and GenPharm International; U.S. Pat. No. 5,545,807 to Surani et al.;International Publication Nos. WO 98/24884, published on Jun. 11, 1998;WO 94/25585, published Nov. 10, 1994; WO 93/1227, published Jun. 24,1993; WO 92/22645, published Dec. 23, 1992; WO 92/03918, published Mar.19, 1992.

Immunizations

To generate fully human antibodies to CD27, transgenic ortranschromosomal mice containing human immunoglobulin genes (e.g.,HCo12, HCo7 or KM mice) can be immunized with a purified or enrichedpreparation of the CD27 antigen and/or cells expressing CD27, asdescribed, for example, by Lonberg et al. (1994) Nature 368(6474):856-859; Fishwild et al. (1996) Nature Biotechnology 14: 845-851 and WO98/24884. As described herein, HuMAb mice are immunized either withrecombinant CD27 proteins or cell lines expressing CD27 as immunogens.Alternatively, mice can be immunized with DNA encoding human CD27.Preferably, the mice will be 6-16 weeks of age upon the first infusion.For example, a purified or enriched preparation (5-50 μg) of therecombinant CD27 antigen can be used to immunize the HuMAb miceintraperitoneally. In the event that immunizations using a purified orenriched preparation of the CD27 antigen do not result in antibodies,mice can also be immunized with cells expressing CD27, e.g., a cellline, to promote immune responses. Exemplary cell lines includeCD27-overexpressing stable CHO and Raji cell lines.

Cumulative experience with various antigens has shown that the HuMAbtransgenic mice respond best when initially immunized intraperitoneally(IP) or subcutaneously (SC) with antigen in complete Freund's adjuvant,followed by every other week IP/SC immunizations (up to a total of 10)with antigen in incomplete Freund's adjuvant. The immune response can bemonitored over the course of the immunization protocol with plasmasamples being obtained by retroorbital bleeds. The plasma can bescreened by ELISA (as described below), and mice with sufficient titersof anti-CD27 human immunoglobulin can be used for fusions. Mice can beboosted intravenously with antigen 3 days before sacrifice and removalof the spleen.

Generation of Hybridomas Producing Monoclonal Antibodies to CD27

To generate hybridomas producing monoclonal antibodies to CD27,splenocytes and lymph node cells from immunized mice can be isolated andfused to an appropriate immortalized cell line, such as a mouse myelomacell line. The resulting hybridomas can then be screened for theproduction of antigen-specific antibodies. For example, single cellsuspensions of splenic lymphocytes from immunized mice can be fused toSP2/0-Ag8.653 nonsecreting mouse myeloma cells (ATCC, CRL 1580) with 50%PEG (w/v). Cells can be plated at approximately 1×10⁵ in flat bottommicrotiter plate, followed by a two week incubation in selective mediumcontaining besides usual reagents 10% fetal Clone Serum, 5-10% origenhybridoma cloning factor (IGEN) and 1×HAT (Sigma). After approximatelytwo weeks, cells can be cultured in medium in which the HAT is replacedwith HT. Individual wells can then be screened by ELISA for humananti-CD27 monoclonal IgM and IgG antibodies, or for binding to thesurface of cells expressing CD27, e.g., a CHO cell line expressing CD27,by FLISA (fluorescence-linked immunosorbent assay). Once extensivehybridoma growth occurs, medium can be observed usually after 10-14days. The antibody secreting hybridomas can be replated, screened again,and if still positive for IgG, anti-CD27 monoclonal antibodies can besubcloned at least twice by limiting dilution. The stable subclones canthen be cultured in vitro to generate antibody in tissue culture mediumfor characterization.

Generation of Transfectomas Producing Monoclonal Antibodies to CD27

Antibodies of the invention also can be produced in a host celltransfectoma using, for example, a combination of recombinant DNAtechniques and gene transfection methods as is well known in the art(Morrison, S. (1985) Science 229:1202).

For example, in one embodiment, the gene(s) of interest, e.g., humanantibody genes, can be ligated into an expression vector such as aeukaryotic expression plasmid such as used by GS gene expression systemdisclosed in WO 87/04462, WO 89/01036 and EP 338 841 or other expressionsystems well known in the art. The purified plasmid with the clonedantibody genes can be introduced in eukaryotic host cells such asCHO-cells or NSO-cells or alternatively other eukaryotic cells like aplant derived cells, fungi or yeast cells. The method used to introducethese genes could be methods described in the art such aselectroporation, lipofectine, lipofectamine or other. After introducingthese antibody genes in the host cells, cells expressing the antibodycan be identified and selected. These cells represent the transfectomaswhich can then be amplified for their expression level and upscaled toproduce antibodies. Recombinant antibodies can be isolated and purifiedfrom these culture supernatants and/or cells.

Alternatively these cloned antibody genes can be expressed in otherexpression systems such as E. coli or in complete organisms or can besynthetically expressed.

Use of Partial Antibody Sequences to Express Intact Antibodies

Antibodies interact with target antigens predominantly through aminoacid residues that are located in the six heavy and light chaincomplementarity determining regions (CDRs). For this reason, the aminoacid sequences within CDRs are more diverse between individualantibodies than sequences outside of CDRs. Because CDR sequences areresponsible for most antibody-antigen interactions, it is possible toexpress recombinant antibodies that mimic the properties of specificnaturally occurring antibodies by constructing expression vectors thatinclude CDR sequences from the specific naturally occurring antibodygrafted onto framework sequences from a different antibody withdifferent properties (see, e.g., Riechmann, L. et al., 1998, Nature332:323-327; Jones, P. et al., 1986, Nature 321:522-525; and Queen, C.et al., 1989, Proc. Natl. Acad. See. U.S.A. 86:10029-10033). Suchframework sequences can be obtained from public DNA databases thatinclude germline antibody gene sequences. These germline sequences willdiffer from mature antibody gene sequences because they will not includecompletely assembled variable genes, which are formed by V(D)J joiningduring B cell maturation. Germline gene sequences will also differ fromthe sequences of a high affinity secondary repertoire antibody atindividual evenly across the variable region. For example, somaticmutations are relatively infrequent in the amino-terminal portion offramework region. For example, somatic mutations are relativelyinfrequent in the amino terminal portion of framework region 1 and inthe carboxy-terminal portion of framework region 4. Furthermore, manysomatic mutations do not significantly alter the binding properties ofthe antibody. For this reason, it is not necessary to obtain the entireDNA sequence of a particular antibody in order to recreate an intactrecombinant antibody having binding properties similar to those of theoriginal antibody (see PCT/US99/05535 filed on Mar. 12, 1999). Partialheavy and light chain sequence spanning the CDR regions is typicallysufficient for this purpose. The partial sequence is used to determinewhich germline variable and joining gene segments contributed to therecombined antibody variable genes. The germline sequence is then usedto fill in missing portions of the variable regions. Heavy and lightchain leader sequences are cleaved during protein maturation and do notcontribute to the properties of the final antibody. To add missingsequences, cloned cDNA sequences can be combined with syntheticoligonucleotides by ligation or PCR amplification. Alternatively, theentire variable region can be synthesized as a set of short,overlapping, oligonucleotides and combined by PCR amplification tocreate an entirely synthetic variable region clone. This process hascertain advantages such as elimination or inclusion or particularrestriction sites, or optimization of particular codons.

The nucleotide sequences of heavy and light chain transcripts from ahybridoma are used to design an overlapping set of syntheticoligonucleotides to create synthetic V sequences with identical aminoacid coding capacities as the natural sequences. The synthetic heavy andkappa chain sequences can differ from the natural sequences in threeways: strings of repeated nucleotide bases are interrupted to facilitateoligonucleotide synthesis and PCR amplification; optimal translationinitiation sites are incorporated according to Kozak's rules (Kozak,1991, J. Biol. Chem. 266:19867-19870); and, HindIII sites are engineeredupstream of the translation initiation sites.

For both the heavy and light chain variable regions, the optimizedcoding, and corresponding non-coding, strand sequences are broken downinto 30-50 nucleotide approximately the midpoint of the correspondingnon-coding oligonucleotide. Thus, for each chain, the oligonucleotidescan be assembled into overlapping double stranded sets that spansegments of 150-400 nucleotides. The pools are then used as templates toproduce PCR amplification products of 150-400 nucleotides. Typically, asingle variable region oligonucleotide set will be broken down into twopools which are separately amplified to generate two overlapping PCRproducts. These overlapping products are then combined by PCRamplification to form the complete variable region. It may also bedesirable to include an overlapping fragment of the heavy or light chainconstant region (including the BbsI site of the kappa light chain, orthe AgeI site if the gamma heavy chain) in the PCR amplification togenerate fragments that can easily be cloned into the expression vectorconstructs.

The reconstructed heavy and light chain variable regions are thencombined with cloned promoter, leader sequence, translation initiation,leader sequence, constant region, 3′ untranslated, polyadenylation, andtranscription termination, sequences to form expression vectorconstructs. The heavy and light chain expression constructs can becombined into a single vector, co-transfected, serially transfected, orseparately transfected into host cells which are then fused to form ahost cell expressing both chains.

Plasmids for use in construction of expression vectors were constructedso that PCR amplified V heavy and V kappa light chain cDNA sequencescould be used to reconstruct complete heavy and light chain minigenes.These plasmids can be used to express completely human IgG₁κ or IgG₄κantibodies. Fully human and chimeric antibodies of the present inventionalso include IgG2, IgG3, IgE, IgA, IgM, and IgD antibodies. Similarplasmids can be constructed for expression of other heavy chainisotypes, or for expression of antibodies comprising lambda lightchains.

Thus, in another aspect of the invention, structural features ofanti-CD27 antibodies of the invention are used to create structurallyrelated anti-CD27 antibodies that retain at least one functionalproperty of the antibodies of the invention, such as, for example,

(1) inhibits (e.g., completely or partially blocks) binding of CD70 toCD27 expressing cells by at least about 70% (e.g., by at least about70%, or by at least 70%, at an antibody concentration of 10 μg/ml);

(2) binds to human CD27 with an equilibrium dissociation constant Kd of10⁻⁹ M or less, or alternatively, an equilibrium association constant Kaof 10⁺⁹ M⁻¹ or greater

(3) induces at least about 30% complement mediated cytotoxicity (CDC) ofCD27 expressing cells at a concentration of 10 μg/ml (or induces atleast 30%, or at least about 40% or at least 40% CDC of CD27 expressingcells at a concentration of 10 μg/ml);

(4) induces at least about 30% specific ADCC-mediated lysis of CD27expressing cells at a concentration of 10 μg/ml (or induces at least30%, or at least about 40% or at least 40% specific ADCC-mediated lysisof CD27 expressing cells at a concentration of 10 μg/ml);(5) prevents or inhibits the growth of CD27-expressing tumor cells in axenograft model (e.g., reduces tumor size in severe combinedimmunodeficiency (SCID) mice by at least about 50% 20-days post tumorcell inoculation in vivo at 0.5 mg ip on at least 6 days);(6) induces or enhances antigen specific immune responses when combinedwith a vaccine or other antigen;(7) induces or enhances immune responses, in particular but not limitedto TH1 immune responses;(8) induces or enhances T-cell activity, in particular but not limitedto specific CD8+ T-cell numbers or functional activity, or T cellproliferation or activation; and/or(9) reduces or inhibits T cell proliferation or activation. In oneembodiment, one or more CDR regions of antibodies of the invention canbe combined recombinantly with known framework regions and CDRs tocreate additional, recombinantly-engineered, anti-CD27 antibodies of theinvention. The heavy and light chain variable framework regions can bederived from the same or different antibody sequences. The antibodysequences can be the sequences of naturally occurring antibodies or canbe consensus sequences of several antibodies. See Kettleborough et al.,Protein Engineering 4:773 (1991); Kolbinger et al., Protein Engineering6:971 (1993) and Carter et al., WO 92/22653.

Accordingly, in another embodiment, the invention provides a method forpreparing an anti-CD27 antibody including: preparing an antibodyincluding (1) heavy chain framework regions and heavy chain CDRs, whereat least one of the heavy chain CDRs includes an amino acid sequenceselected from the amino acid sequences of CDRs shown in SEQ ID NOs: 8,9, 10, 26, 27, 28, 38, 39, 40, 50, 51, 52, 62, 63, 64, 74, 75, 76, 86,87, 88, 104, 105, 106; and (2) light chain framework regions and lightchain CDRs, where at least one of the light chain CDRs includes an aminoacid sequence selected from the amino acid sequences of CDRs shown inSEQ ID NOs: SEQ ID NOs: 14, 15, 16, 20, 21, 22, 32, 33, 34, 44, 45, 46,56, 57, 58, 68, 69, 70, 80, 81, 82, 92, 93, 94, 98, 99, 100, 110, 111,112; where the antibody retains the ability to bind to CD27. The abilityof the antibody to bind CD27 can be determined using standard bindingassays, such as those set forth in the Examples (e.g., an ELISA or aFLISA).

It is well known in the art that antibody heavy and light chain CDR3domains play a particularly important role in the bindingspecificity/affinity of an antibody for an antigen (see, Hall et al., J.Imunol., 149:1605-1612 (1992); Polymenis et al., J. Immunol.,152:5318-5329 (1994); Jahn et al., Immunobiol., 193:400-419 (1995);Klimka et al., Brit. J. Cancer, 83:252-260 (2000); Beiboer et al., J.Mol. Biol, 296:833-849 (2000); Rader et al., Proc. Natl. Acad. Sci. USA,95:8910-8915 (1998); Barbas et al., J. Am. Chem. Soc., 116:2161-2162(1994); Ditzel et al., J. Immunol., 157:739-749 (1996)). Accordingly,the recombinant antibodies of the invention prepared as set forth abovepreferably comprise the heavy and/or light chain CDR3s of antibodies1F5, 1H8, 3H12, 3A10, 2C2, 2G9, 3H8, and 1G5. The antibodies further cancomprise the CDR2s of antibodies 1F5, 1H8, 3H12, 3A10, 2C2, 2G9, 3H8,and 1G5. The antibodies further can comprise the CDR1s of antibodies1F5, 1H8, 3H12, 3A10, 2C2, 2G9, 3H8, and 1G5. The antibodies can furthercomprise any combinations of the CDRs.

Accordingly, in another embodiment, the invention further providesanti-CD27 antibodies comprising: (1) heavy chain framework regions, aheavy chain CDR1 region, a heavy chain CDR2 region, and a heavy chainCDR3 region, wherein the heavy chain CDR3 region is selected from theCDR3s of 1F5, 1H8, 3H12, 3A10, 2C2, 2G9, 3H8, and 1G5 and (2) lightchain framework regions, a light chain CDR1 region, a light chain CDR2region, and a light chain CDR3 region, wherein the light chain CDR3region is selected from the CDR3s of 1F5, 1H8, 3H12, 3A10, 2C2, 2G9,3H8, and 1G5, wherein the antibody binds CD27. The antibody may furtherinclude the heavy chain CDR2 and/or the light chain CDR2 of antibodies1F5, 1H8, 3H12, 3A10, 2C2, 2G9, 3H8, and 1G5. The antibody may furthercomprise the heavy chain CDR1 and/or the light chain CDR1 of antibodies1F5, 1H8, 3H12, 3A10, 2C2, 2G9, 3H8, and 1G5.

Generation of Antibodies Having Modified Sequences

In another embodiment, the variable region sequences, or portionsthereof, of the anti-CD27 antibodies of the invention are modified tocreate structurally related anti-CD27 antibodies that retain binding(i.e., to the same epitope as the unmodified antibody) and, thus, arefunctionally equivalent. Methods for identifying residues that can bealtered without removing antigen binding are well-known in the art (see,e.g., Marks et al. (Biotechnology (1992) 10(7):779-83 (monoclonalantibodies diversification by shuffling light chain variable regions,then heavy chain variable regions with fixed CDR3 sequence changes),Jespers et al. (1994) Biotechnology 12(9):899-903 (selection of humanantibodies from phage display repertoires to a single epitope of anantigen), Sharon et al. (1986) PNAS USA 83(8):2628-31 (site-directedmutagenesis of an invariant amino acid residue at the variable-diversitysegments junction of an antibody); Casson et al. (1995) J. Immunol.155(12):5647-54 (evolution of loss and change of specificity resultingfrom random mutagenesis of an antibody heavy chain variable region).

Accordingly, in one aspect of the invention, the CDR1, 2, and/or 3regions of the engineered antibodies described above can comprise theexact amino acid sequence(s) as those of antibodies 1F5, 1H8, 3H12,3A10, 2C2, 2G9, 3H8, and 1G5 disclosed herein. However, in other aspectsof the invention, the antibodies comprise derivatives from the exact CDRsequences of 1F5, 1H8, 3H12, 3A10, 2C2, 2G9, 3H8, and 1G5 yet stillretain the ability of to bind CD27 effectively. Such sequencemodifications may include one or more amino acid additions, deletions,or substitutions, e.g., conservative sequence modifications as describedabove. Sequence modifications may also be based on the consensussequences described above for the particular CDR1, CDR2, and CDR3sequences of antibodies 1F5, 1H8, 3H12, 3A10, 2C2, 2G9, 3H8, and 1G5.

Accordingly, in another embodiment, the engineered antibody may becomposed of one or more CDRs that are, for example, 90%, 95%, 98% or99.5% identical to one or more CDRs of antibodies 1F5, 1H8, 3H12, 3A10,2C2, 2G9, 3H8, and 1G5. Ranges intermediate to the above-recited values,e.g., CDRs that are 90-95%, 95-98%, or 98-100% identical identity to oneor more of the above sequences are also intended to be encompassed bythe present invention.

In another embodiment, one or more residues of a CDR may be altered tomodify binding to achieve a more favored on-rate of binding, a morefavored off-rate of binding, or both, such that an idealized bindingconstant is achieved. Using this strategy, an antibody having ultra highbinding affinity of, for example, 10¹⁰ M⁻¹ or more, can be achieved.Affinity maturation techniques, well known in the art and thosedescribed herein, can be used to alter the CDR region(s) followed byscreening of the resultant binding molecules for the desired change inbinding. Accordingly, as CDR(s) are altered, changes in binding affinityas well as immunogenicity can be monitored and scored such that anantibody optimized for the best combined binding and low immunogenicityare achieved.

In addition to or instead of modifications within the CDRs,modifications can also be made within one or more of the frameworkregions, FR1, FR2, FR3 and FR4, of the heavy and/or the light chainvariable regions of a antibody, so long as these modifications do noteliminate the binding affinity of the antibody. For example, one or morenon-germline amino acid residues in the framework regions of the heavyand/or the light chain variable region of a antibody of the invention,is substituted with a germline amino acid residue, i.e., thecorresponding amino acid residue in the human germline sequence for theheavy or the light chain variable region, which the antibody hassignificant sequence identity with. For example, an antibody chain canbe aligned to a germline antibody chain which it shares significantsequence identity with, and the amino acid residues which do not matchbetween antibody framework sequence and the germline chain framework canbe substituted with corresponding residues from the germline sequence.When an amino acid differs between a antibody variable framework regionand an equivalent human germline sequence variable framework region, theantibody framework amino acid should usually be substituted by theequivalent human germline sequence amino acid if it is reasonablyexpected that the amino acid falls within one of the followingcategories:

(1) an amino acid residue which noncovalently binds antigen directly,

(2) an amino acid residue which is adjacent to a CDR region,

(3) an amino acid residue which otherwise interacts with a CDR region(e.g., is within about 3-6 Å of a CDR region as determined by computermodeling), or

(4) an amino acid reside which participates in the VL-VH interface.

Residues which “noncovalently bind antigen directly” include amino acidsin positions in framework regions which have a good probability ofdirectly interacting with amino acids on the antigen according toestablished chemical forces, for example, by hydrogen bonding, Van derWaals forces, hydrophobic interactions, and the like. Accordingly, inone embodiment, an amino acid residue in the framework region of aantibody of the invention is substituted with the corresponding germlineamino acid residue which noncovalently binds antigen directly.

Residues which are “adjacent to a CDR region” include amino acidresidues in positions immediately adjacent to one or more of the CDRs inthe primary sequence of the antibody, for example, in positionsimmediately adjacent to a CDR as defined by Kabat, or a CDR as definedby Chothia (see e.g., Chothia and Lesk J. Mol. Biol. 196:901 (1987)).Accordingly, in one embodiment, an amino acid residue within theframework region of an antibody of the invention is substituted with acorresponding germline amino acid residue which is adjacent to a CDRregion.

Residues that “otherwise interact with a CDR region” include those thatare determined by secondary structural analysis to be in a spatialorientation sufficient to affect a CDR region. Such amino acids willgenerally have a side chain atom within about 3 angstrom units (Å) ofsome atom in the CDRs and must contain an atom that could interact withthe CDR atoms according to established chemical forces, such as thoselisted above. Accordingly, in one embodiment, an amino acid residuewithin the framework region of an antibody of the invention issubstituted with the corresponding germline amino acid residue whichotherwise interacts with a CDR region.

The amino acids at several positions in the framework are known to beimportant for determining CDR confirmation (e.g., capable of interactingwith the CDRs) in many antibodies (Chothia and Lesk, supra, Chothia etal., supra and Tramontano et al., J. Mol. Biol. 215:175 (1990), all ofwhich are incorporated herein by reference). These authors identifiedconserved framework residues important for CDR conformation by analysisof the structures of several known antibodies. The antibodies analyzedfell into a limited number of structural or “canonical” classes based onthe conformation of the CDRs. Conserved framework residues withinmembers of a canonical class are referred to as “canonical” residues.Canonical residues include residues 2, 25, 29, 30, 33, 48, 64, 71, 90,94 and 95 of the light chain and residues 24, 26, 29, 34, 54, 55, 71 and94 of the heavy chain. Additional residues (e.g., CDRstructure-determining residues) can be identified according to themethodology of Martin and Thorton (1996) J. Mol. Biol. 263:800. Notably,the amino acids at positions 2, 48, 64 and 71 of the light chain and26-30, 71 and 94 of the heavy chain (numbering according to Kabat) areknown to be capable of interacting with the CDRs in many antibodies. Theamino acids at positions 35 in the light chain and 93 and 103 in theheavy chain are also likely to interact with the CDRs. Additionalresidues which may effect conformation of the CDRs can be identifiedaccording to the methodology of Foote and Winter (1992) J. Mol. Biol.224:487. Such residues are termed “vernier” residues and are thoseresidues in the framework region closely underlying (i.e., forming a“platform” under) the CDRs.

Residues which “participate in the VL-VH interface” or “packingresidues” include those residues at the interface between VL and VH asdefined, for example, by Novotny and Haber, Proc. Natl. Acad. Sci. USA,82:4592-66 (1985) or Chothia et al, supra.

Occasionally, there is some ambiguity about whether a particular aminoacid falls within one or more of the above-mentioned categories. In suchinstances, alternative variant antibodies are produced, one of which hasthat particular substitution, the other of which does not. Alternativevariant antibodies so produced can be tested in any of the assaysdescribed herein for the desired activity, and the preferred antibodyselected.

Additional candidates for substitution within the framework region areamino acids that are unusual or “rare” for an antibody at that position.These amino acids can be substituted with amino acids from theequivalent position of the human germline sequence or from theequivalent positions of more typical antibodies. For example,substitution may be desirable when the amino acid in a framework regionof the antibody is rare for that position and the corresponding aminoacid in the germline sequence is common for that position inimmunoglobulin sequences; or when the amino acid in the antibody is rarefor that position and the corresponding amino acid in the germlinesequence is also rare, relative to other sequences. It is contemplatedthat by replacing an unusual amino acid with an amino acid from thegermline sequence that happens to be typical for antibodies, theantibody may be made less immunogenic.

The term “rare”, as used herein, indicates an amino acid occurring atthat position in less than about 20%, preferably less than about 10%,more preferably less than about 5%, even more preferably less than about3%, even more preferably less than about 2% and even more preferablyless than about 1% of sequences in a representative sample of sequences,and the term “common”, as used herein, indicates an amino acid occurringin more than about 25% but usually more than about 50% of sequences in arepresentative sample. For example, all light and heavy chain variableregion sequences are respectively grouped into “subgroups” of sequencesthat are especially homologous to each other and have the same aminoacids at certain critical positions (Kabat et al., supra). When decidingwhether an amino acid in an antibody sequence is “rare” or “common”among sequences, it will often be preferable to consider only thosesequences in the same subgroup as the antibody sequence.

In general, the framework regions of antibodies are usuallysubstantially identical, and more usually, identical to the frameworkregions of the human germline sequences from which they were derived. Ofcourse, many of the amino acids in the framework region make little orno direct contribution to the specificity or affinity of an antibody.Thus, many individual conservative substitutions of framework residuescan be tolerated without appreciable change of the specificity oraffinity of the resulting immunoglobulin. Thus, in one embodiment thevariable framework region of the antibody shares at least 85% sequenceidentity to a human germline variable framework region sequence orconsensus of such sequences. In another embodiment, the variableframework region of the antibody shares at least 90%, 95%, 96%, 97%, 98%or 99% sequence identity to a human germline variable framework regionsequence or consensus of such sequences.

In addition to simply binding CD27, an antibody may be selected for itsretention of other functional properties of antibodies of the invention,such as, for example:

(1) inhibits (e.g., completely or partially blocks) binding of CD70 toCD27 expressing cells by at least about 70%;

(2) binds to human CD27 with an equilibrium dissociation constant Kd of10⁻⁹ M or less, or alternatively, an equilibrium association constant Kaof 10⁺⁹ M⁻¹ or greater;

(3) induces at least about 40% complement mediated cytotoxicity (CDC) ofCD27 expressing cells at a concentration of 10 μg/ml; and/or

(4) induces at least about 40% ADCC mediated specific lysis of CD27expressing cells at a concentration of 10 μg/ml.

Characterization of Monoclonal Antibodies to CD27

Monoclonal antibodies of the invention can be characterized for bindingto CD27 using a variety of known techniques. Generally, the antibodiesare initially characterized by ELISA. Briefly, microtiter plates can becoated with purified CD27 in PBS, and then blocked with irrelevantproteins such as bovine serum albumin (BSA) diluted in PBS. Dilutions ofplasma from CD27-immunized mice are added to each well and incubated for1-2 hours at 37° C. The plates are washed with PBS/Tween 20 and thenincubated with a goat-anti-human IgG Fc-specific polyclonal reagentconjugated to alkaline phosphatase for 1 hour at 37° C. After washing,the plates are developed with ABTS substrate, and analyzed at OD of 405.Preferably, mice which develop the highest titers will be used forfusions.

An ELISA assay as described above can be used to screen for antibodiesand, thus, hybridomas that produce antibodies that show positivereactivity with the CD27 immunogen. Hybridomas that bind, preferablywith high affinity, to CD27 can then be subcloned and furthercharacterized. One clone from each hybridoma, which retains thereactivity of the parent cells (by ELISA), can then be chosen for makinga cell bank, and for antibody purification.

To purify anti-CD27 antibodies, selected hybridomas can be grown inroller bottles, two-liter spinner-flasks or other culture systems.Supernatants can be filtered and concentrated before affinitychromatography with protein A-Sepharose (Pharmacia, Piscataway, N.J.) topurify the protein. After buffer exchange to PBS, the concentration canbe determined by OD₂₈₀ using 1.43 extinction coefficient or preferablyby nephelometric analysis. IgG can be checked by gel electrophoresis andby antigen specific method.

To determine if the selected anti-CD27 monoclonal antibodies bind tounique epitopes, each antibody can be biotinylated using commerciallyavailable reagents (Pierce, Rockford, Ill.). Biotinylated MAb bindingcan be detected with a streptavidin labeled probe. To determine theisotype of purified antibodies, isotype ELISAs can be performed usingart recognized techniques. For example, wells of microtiter plates canbe coated with 10 μg/ml of anti-Ig overnight at 4° C. After blockingwith 5% BSA, the plates are reacted with 10 μg/ml of monoclonalantibodies or purified isotype controls, at ambient temperature for twohours. The wells can then be reacted with either IgG1 or other isotypespecific conjugated probes. Plates are developed and analyzed asdescribed above.

To test the binding of monoclonal antibodies to live cells expressingCD27, flow cytometry can be used. Briefly, cell lines and/or human PBMCsexpressing membrane-bound CD27 (grown under standard growth conditions)are mixed with various concentrations of monoclonal antibodies in PBScontaining 0.1% BSA at 4° C. for 1 hour. After washing, the cells arereacted with Fluorescein-labeled anti-IgG antibody under the sameconditions as the primary antibody staining. The samples can be analyzedby FACScan instrument using light and side scatter properties to gate onsingle cells and binding of the labeled antibodies is determined. Analternative assay using fluorescence microscopy may be used (in additionto or instead of) the flow cytometry assay. Cells can be stained exactlyas described above and examined by fluorescence microscopy. This methodallows visualization of individual cells, but may have diminishedsensitivity depending on the density of the antigen.

Anti-CD27 IgGs can be further tested for reactivity with the CD27antigen by Western blotting. Briefly, cell extracts from cellsexpressing CD27 can be prepared and subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis. After electrophoresis, the separatedantigens will be transferred to nitrocellulose membranes, blocked with20% mouse serum, and probed with the monoclonal antibodies to be tested.IgG binding can be detected using anti-IgG alkaline phosphatase anddeveloped with BCIP/NBT substrate tablets (Sigma Chem. Co., St. Louis,Mo.).

Methods for analyzing binding affinity, cross-reactivity, and bindingkinetics of various anti-CD27 antibodies include standard assays knownin the art, for example, Biacore™ surface plasmon resonance (SPR)analysis using a Biacore™ 2000 SPR instrument (Biacore AB, Uppsala,Sweden), as described in Example 2 herein.

II Immunotoxins

In another embodiment, the antibodies of the present invention arelinked to a therapeutic moiety, such as a cytotoxin, a drug or aradioisotope. When conjugated to a cytotoxin, these antibody conjugatesare referred to as “immunotoxins.” A cytotoxin or cytotoxic agentincludes any agent that is detrimental to (e.g., kills) cells. Examplesinclude taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin,doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, and puromycin and analogsor homologs thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine). An antibody of the presentinvention can be conjugated to a radioisotope, e.g., radioactive iodine,to generate cytotoxic radiopharmaceuticals for treating adendritic-related disorder, such as an autoimmune or inflammatorydisease, or graft versus host disease.

The antibody conjugates of the invention can be used to modify a givenbiological response, and the drug moiety is not to be construed aslimited to classical chemical therapeutic agents. For example, the drugmoiety may be a protein or polypeptide possessing a desired biologicalactivity. Such proteins may include, for example, an enzymaticallyactive toxin, or active fragment thereof, such as abrin, ricin A,pseudomonas exotoxin, or diphtheria toxin; a protein such as tumornecrosis factor or interferon-γ; or, biological response modifiers suchas, for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev., 62:119-58 (1982).

III. Compositions

In another embodiment, the present invention provides a composition,e.g., a composition, containing one or a combination of monoclonalantibodies of the present invention, formulated together with a carrier(e.g., a pharmaceutically acceptable carrier). Compositions containingbispecific molecules which comprise an antibody of the present inventionare also provided. In one embodiment, the compositions include acombination of multiple (e.g., two or more) isolated antibodies of theinvention. Preferably, each of the antibodies of the composition bindsto a distinct, pre-selected epitope of CD27.

Pharmaceutical compositions of the invention also can be administered incombination therapy, i.e., combined with other agents. For example, thecombination therapy can include a composition of the present inventionwith at least one or more additional therapeutic agents, such asanti-inflammatory agents, DMARDs (disease-modifying anti-rheumaticdrugs), immunosuppressive agents, and chemotherapeutics. Thepharmaceutical compositions of the invention can also be administered inconjunction with radiation therapy. Co-administration with otherantibodies is also encompassed by the invention.

As used herein, the terms “carrier” and “pharmaceutically acceptablecarrier” includes any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like that are physiologically compatible. Preferably,the carrier is suitable for intravenous, intramuscular, subcutaneous,parenteral, spinal or epidermal administration (e.g., by injection orinfusion). Depending on the route of administration, the activecompound, i.e., antibody, bispecific and multispecific molecule, may becoated in a material to protect the compound from the action of acidsand other natural conditions that may inactivate the compound.

Examples of adjuvants which may be used with the antibodies andconstructs of the present invention include: Freund's IncompleteAdjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Mich.);Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.); AS-2(SmithKline Beecham, Philadelphia, Pa.); aluminum salts such as aluminumhydroxide gel (alum) or aluminum phosphate; salts of calcium, iron orzinc; an insoluble suspension of acylated tyrosine; acylated sugars;cationically or anionically derivatised polysaccharides;polyphosphazenes; biodegradable microspheres; cytokines, such as GM-CSF,interleukin-2, -7, -12, and other like factors; 3D-MPL; CpGoligonucleotide; and monophosphoryl lipid A, for example 3-de-O-acylatedmonophosphoryl lipid A.

MPL adjuvants are available from Corixa Corporation (Seattle, Wash.;see, for example, U.S. Pat. Nos. 4,436,727; 4,877,611; 4,866,034 and4,912,094). CpG-containing oligonucleotides (in which the CpGdinucleotide is unmethylated) are well known and are described, forexample, in WO 96/02555, WO 99/33488 and U.S. Pat. Nos. 6,008,200 and5,856,462 Immunostimulatory DNA sequences are also described, forexample, by Sato et al., Science 273:352, 1996.

Further alternative adjuvants include, for example, saponins, such asQuil A, or derivatives thereof, including QS21 and QS7 (AquilaBiopharmaceuticals Inc., Framingham, Mass.); Escin; Digitonin; orGypsophila or Chenopodium quinoa saponins; Montanide ISA 720 (Seppic,France); SAF (Chiron, Calif., United States); ISCOMS (CSL), MF-59(Chiron); the SBAS series of adjuvants (e.g., SBAS-2 or SBAS-4,available from SmithKline Beecham, Rixensart, Belgium); Detox(Enhanzyn™) (Corixa, Hamilton, Mont.); RC-529 (Corixa, Hamilton, Mont.)and other aminoalkyl glucosaminide 4-phosphates (AGPs); polyoxyethyleneether adjuvants such as those described in WO 99/52549A1; syntheticimidazoquinolines such as imiquimod [S-26308, R-837], (Harrison, et al.,Vaccine 19: 1820-1826, 2001; and resiquimod [S-28463, R-848] (Vasilakos,et al., Cellular immunology 204: 64-74, 2000; Schiff bases of carbonylsand amines that are constitutively expressed on antigen presenting celland T-cell surfaces, such as tucaresol (Rhodes, J. et al., Nature 377:71-75, 1995); cytokine, chemokine and co-stimulatory molecules as eitherprotein or peptide, including for example pro-inflammatory cytokinessuch as Interferon, GM-CSF, IL-1 alpha, IL-1 beta, TGF-alpha andTGF-beta, Th1 inducers such as interferon gamma, IL-2, IL-12, IL-15,IL-18 and IL-21, Th2 inducers such as IL-4, IL-5, IL-6, IL-10 and IL-13and other chemokine and co-stimulatory genes such as MCP-1, MIP-1 alpha,MIP-1 beta, RANTES, TCA-3, CD80, CD86 and CD40L; immunostimulatoryagents targeting ligands such as CTLA-4 and L-selectin, apoptosisstimulating proteins and peptides such as Fas; synthetic lipid basedadjuvants, such as vaxfectin, (Reyes et al., Vaccine 19: 3778-3786,2001) squalene, alpha-tocopherol, polysorbate 80, DOPC and cholesterol;endotoxin, [LPS], (Beutler, B., Current Opinion in Microbiology 3:23-30, 2000); ligands that trigger Toll receptors to produceTh1-inducing cytokines, such as synthetic Mycobacterial lipoproteins,Mycobacterial protein p19, peptidoglycan, teichoic acid and lipid A; andCT (cholera toxin, subunits A and B) and LT (heat labile enterotoxinfrom E. coli, subunits A and B), heat shock protein family (HSPs), andLLO (listeriolysin O; WO 01/72329). These and various further Toll-likeReceptor (TLR) agonists are described for example in Kanzler et al,Nature Medicine, May 2007, Vol 13, No 5. A preferred immunostimulatoryagent for use in combination with an anti-CD27 antibody of the inventionis a TLR3 agonist, such as Poly IC.

A “pharmaceutically acceptable salt” refers to a salt that retains thedesired biological activity of the parent compound and does not impartany undesired toxicological effects (see e.g., Berge, S. M., et al.(1977) J. Pharm. Sci. 66:1-19). Examples of such salts include acidaddition salts and base addition salts. Acid addition salts includethose derived from nontoxic inorganic acids, such as hydrochloric,nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous andthe like, as well as from nontoxic organic acids such as aliphatic mono-and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acidsand the like. Base addition salts include those derived from alkalineearth metals, such as sodium, potassium, magnesium, calcium and thelike, as well as from nontoxic organic amines, such asN,N′-dibenzylethylenediamine, N-methylglucamine, chloroprocaine,choline, diethanolamine, ethylenediamine, procaine and the like.

A composition of the present invention can be administered by a varietyof methods known in the art. As will be appreciated by the skilledartisan, the route and/or mode of administration will vary dependingupon the desired results. The active compounds can be prepared withcarriers that will protect the compound against rapid release, such as acontrolled release formulation, including implants, transdermal patches,and microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. See, e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978.

To administer a compound of the invention by certain routes ofadministration, it may be necessary to coat the compound with, orco-administer the compound with, a material to prevent its inactivation.For example, the compound may be administered to a subject in anappropriate carrier, for example, liposomes, or a diluent. Acceptablediluents include saline and aqueous buffer solutions. Liposomes includewater-in-oil-in-water CGF emulsions as well as conventional liposomes(Strejan et al. (1984) J. Neuroimmunol. 7:27).

Carriers include sterile aqueous solutions or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. The use of such media and agents forpharmaceutically active substances is known in the art. Except insofaras any conventional media or agent is incompatible with the activecompound, use thereof in the pharmaceutical compositions of theinvention is contemplated. Supplementary active compounds can also beincorporated into the compositions.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, or sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. For example, the antibodies ofthe invention may be administered once or twice weekly by subcutaneousor intramuscular injection or once or twice monthly by subcutaneous orintramuscular injection.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subjects to be treated; each unitcontains a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on (a) the uniquecharacteristics of the active compound and the particular therapeuticeffect to be achieved, and (b) the limitations inherent in the art ofcompounding such an active compound for the treatment of sensitivity inindividuals.

Examples of pharmaceutically-acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

For the therapeutic compositions, formulations of the present inventioninclude those suitable for oral, nasal, topical (including buccal andsublingual), rectal, vaginal and/or parenteral administration. Theformulations may conveniently be presented in unit dosage form and maybe prepared by any methods known in the art of pharmacy. The amount ofactive ingredient which can be combined with a carrier material toproduce a single dosage form will vary depending upon the subject beingtreated, and the particular mode of administration. The amount of activeingredient which can be combined with a carrier material to produce asingle dosage form will generally be that amount of the compositionwhich produces a therapeutic effect. Generally, out of one hundredpercent, this amount will range from about 0.001 percent to about ninetypercent of active ingredient, preferably from about 0.005 percent toabout 70 percent, most preferably from about 0.01 percent to about 30percent.

Formulations of the present invention which are suitable for vaginaladministration also include pessaries, tampons, creams, gels, pastes,foams or spray formulations containing such carriers as are known in theart to be appropriate. Dosage forms for the topical or transdermaladministration of compositions of this invention include powders,sprays, ointments, pastes, creams, lotions, gels, solutions, patches andinhalants. The active compound may be mixed under sterile conditionswith a pharmaceutically acceptable carrier, and with any preservatives,buffers, or propellants which may be required.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural and intrasternal injection andinfusion.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofpresence of microorganisms may be ensured both by sterilizationprocedures, supra, and by the inclusion of various antibacterial andantifungal agents, for example, paraben, chlorobutanol, phenol sorbicacid, and the like. It may also be desirable to include isotonic agents,such as sugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas aluminum monostearate and gelatin.

When the compounds of the present invention are administered aspharmaceuticals, to humans and animals, they can be given alone or as apharmaceutical composition containing, for example, 0.001 to 90% (morepreferably, 0.005 to 70%, such as 0.01 to 30%) of active ingredient incombination with a pharmaceutically acceptable carrier.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically acceptable dosage forms by conventional methodsknown to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts. A physician orveterinarian having ordinary skill in the art can readily determine andprescribe the effective amount of the pharmaceutical compositionrequired. For example, the physician or veterinarian could start dosesof the compounds of the invention employed in the pharmaceuticalcomposition at levels lower than that required in order to achieve thedesired therapeutic effect and gradually increase the dosage until thedesired effect is achieved. In general, a suitable daily dose of acomposition of the invention will be that amount of the compound whichis the lowest dose effective to produce a therapeutic effect. Such aneffective dose will generally depend upon the factors described above.It is preferred that administration be intravenous, intramuscular,intraperitoneal, or subcutaneous, preferably administered proximal tothe site of the target. If desired, the effective daily dose of atherapeutic composition may be administered as two, three, four, five,six or more sub-doses administered separately at appropriate intervalsthroughout the day, optionally, in unit dosage forms. While it ispossible for a compound of the present invention to be administeredalone, it is preferable to administer the compound as a pharmaceuticalformulation (composition).

Therapeutic compositions can be administered with medical devices knownin the art. For example, in a preferred embodiment, a therapeuticcomposition of the invention can be administered with a needlelesshypodermic injection device, such as the devices disclosed in U.S. Pat.Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824,or 4,596,556. Examples of well-known implants and modules useful in thepresent invention include: U.S. Pat. No. 4,487,603, which discloses animplantable micro-infusion pump for dispensing medication at acontrolled rate; U.S. Pat. No. 4,486,194, which discloses a therapeuticdevice for administering medicants through the skin; U.S. Pat. No.4,447,233, which discloses a medication infusion pump for deliveringmedication at a precise infusion rate; U.S. Pat. No. 4,447,224, whichdiscloses a variable flow implantable infusion apparatus for continuousdrug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drugdelivery system having multi-chamber compartments; and U.S. Pat. No.4,475,196, which discloses an osmotic drug delivery system. Many othersuch implants, delivery systems, and modules are known to those skilledin the art.

In certain embodiments, the antibodies of the invention can beformulated to ensure proper distribution in vivo. For example, theblood-brain barrier (BBB) excludes many highly hydrophilic compounds. Toensure that the therapeutic compounds of the invention cross the BBB (ifdesired), they can be formulated, for example, in liposomes. For methodsof manufacturing liposomes, see, e.g., U.S. Pat. Nos. 4,522,811;5,374,548; and 5,399,331. The liposomes may comprise one or moremoieties which are selectively transported into specific cells ororgans, thus enhance targeted drug delivery (see, e.g., V. V. Ranade(1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties includefolate or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et al.);mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun.153:1038); antibodies (P. G. Bloeman et al. (1995) FEBS Lett. 357:140;M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180); surfactantprotein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233:134),different species of which may comprise the formulations of theinventions, as well as components of the invented molecules; p120(Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K. Keinanen;M. L. Laukkanen (1994) FEBS Lett. 346:123; J. J. Killion; I. J. Fidler(1994) Immunomethods 4:273. In one embodiment of the invention, thetherapeutic compounds of the invention are formulated in liposomes; in amore preferred embodiment, the liposomes include a targeting moiety. Ina most preferred embodiment, the therapeutic compounds in the liposomesare delivered by bolus injection to a site proximal to the tumor orinfection. The composition must be fluid to the extent that easysyringability exists. It must be stable under the conditions ofmanufacture and storage and must be preserved against the contaminatingaction of microorganisms such as bacteria and fungi.

The ability of a compound to inhibit cancer can be evaluated in ananimal model system predictive of efficacy in human tumors.Alternatively, this property of a composition can be evaluated byexamining the ability of the compound to inhibit, such inhibition invitro by assays known to the skilled practitioner. A therapeuticallyeffective amount of a therapeutic compound can decrease tumor size, orotherwise ameliorate symptoms in a subject. One of ordinary skill in theart would be able to determine such amounts based on such factors as thesubject's size, the severity of the subject's symptoms, and theparticular composition or route of administration selected.

The composition must be sterile and fluid to the extent that thecomposition is deliverable by syringe. In addition to water, the carriercan be an isotonic buffered saline solution, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyetheylene glycol,and the like), and suitable mixtures thereof. Proper fluidity can bemaintained, for example, by use of coating such as lecithin, bymaintenance of required particle size in the case of dispersion and byuse of surfactants. In many cases, it is preferable to include isotonicagents, for example, sugars, polyalcohols such as mannitol or sorbitol,and sodium chloride in the composition. Long-term absorption of theinjectable compositions can be brought about by including in thecomposition an agent which delays absorption, for example, aluminummonostearate or gelatin.

When the active compound is suitably protected, as described above, thecompound may be orally administered, for example, with an inert diluentor an assimilable edible carrier.

IV. Uses and Methods of the Invention

In one embodiment, the antibodies, bispecific molecules, andcompositions of the present invention can be used to treat and/orprevent (e.g., immunize against) a variety of diseases and conditions.

One of the primary disease indications that can be treated is cancer. Inparticular, an anti-CD27 antibody that induces or enhances an immuneresponse can be used in the treatment of cancer. Types of cancersinclude, but are not limited to, leukemia, acute lymphocytic leukemia,acute myelocytic leukemia, myeloblasts promyelocyte myelomonocyticmonocytic erythroleukemia, chronic leukemia, chronic myelocytic(granulocytic) leukemia, chronic lymphocytic leukemia, mantle celllymphoma, primary central nervous system lymphoma, Burkitt's lymphomaand marginal zone B cell lymphoma, Polycythemia vera Lymphoma, Hodgkin'sdisease, non-Hodgkin's disease, multiple myeloma, Waldenstrom'smacroglobulinemia, heavy chain disease, solid tumors, sarcomas, andcarcinomas, fibrosarcoma, myxosarcoma, liposarcoma, chrondrosarcoma,osteogenic sarcoma, osteosarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon sarcoma, colorectal carcinoma, pancreaticcancer, breast cancer, ovarian cancer, prostate cancer, squamous cellcarcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma,sebaceous gland carcinoma, papillary carcinoma, papillaryadenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogeniccarcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma,choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervicalcancer, uterine cancer, testicular tumor, lung carcinoma, small celllung carcinoma, non small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma,retinoblastoma, nasopharyngeal carcinoma, esophageal carcinoma, basalcell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brainand central nervous system (CNS) cancer, cervical cancer,choriocarcinoma, colorectal cancers, connective tissue cancer, cancer ofthe digestive system, endometrial cancer, esophageal cancer, eye cancer,head and neck cancer, gastric cancer, intraepithelial neoplasm, kidneycancer, larynx cancer, liver cancer, lung cancer (small cell, largecell), melanoma, neuroblastoma; oral cavity cancer (for example lip,tongue, mouth and pharynx), ovarian cancer, pancreatic cancer,retinoblastoma, rhabdomyosarcoma, rectal cancer; cancer of therespiratory system, sarcoma, skin cancer, stomach cancer, testicularcancer, thyroid cancer, uterine cancer, and cancer of the urinarysystem. Preferred cancers include CD27-expressing tumors selected fromthe group consisting of chronic lymphocytic leukemia, mantle celllymphoma, primary central nervous system lymphoma, Burkitt's lymphomaand marginal zone B cell lymphoma.

Other disease indications for use of an anti-CD27 antibody that inducesor enhances an immune response include bacterial, fungal, viral andparasitic infectious diseases. Other disease indications for use of ananti-CD27 antibody that inhibits or reduces an immune response includegraft rejection, allergy and autoimmune diseases.

Exemplary autoimmune diseases include, but are not limited to, multiplesclerosis, rheumatoid arthritis, type 1 diabetes, psoriasis, Crohn'sdisease and other inflammatory bowel diseases such as ulcerativecolitis, systemic lupus eythematosus (SLE), autoimmuneencephalomyelitis, myasthenia gravis (MG), Hashimoto's thyroiditis,Goodpasture's syndrome, pemphigus, Graves disease, autoimmune hemolyticanemia, autoimmune thrombocytopenic purpura, scleroderma withanti-collagen antibodies, mixed connective tissue disease, polypyositis,pernicious anemia, idiopathic Addison's disease, autoimmune associatedinfertility, glomerulonephritis, crescentic glomerulonephritis,proliferative glomerulonephritis, bullous pemphigoid, Sjogren'ssyndrome, psoriatic arthritis, insulin resistance, autoimmune diabetesmellitus, autoimmune hepatitis, autoimmune hemophilia, autoimmunelymphoproliferative syndrome (ALPS), autoimmune hepatitis, autoimmunehemophilia, autoimmune lymphoproliferative syndrome, autoimmuneuveoretinitis, Guillain-Bare syndrome, arteriosclerosis and Alzheimer'sdisease. Exemplary allergic disorders include, but are not limited toallergic conjunctivitis, vernal conjunctivitis, vernalkeratoconjunctivitis, and giant papillary conjunctivitis; nasal allergicdisorders, including allergic rhinitis and sinusitis; otic allergicdisorders, including eustachian tube itching; allergic disorders of theupper and lower airways, including intrinsic and extrinsic asthma;allergic disorders of the skin, including dermatitis, eczema andurticaria; and allergic disorders of the gastrointestinal tract.

In another aspect, an antibody of the invention is administered incombination with a vaccine, to enhance the immune response against thevaccine antigen, for example a tumor antigen (to thereby enhance theimmune response against the tumor) or an antigen from an infectiousdisease pathogen (to thereby enhance the immune response against theinfectious disease pathogen). Accordingly, in this embodiment, a vaccineantigen can comprise, for example, an antigen or antigenic compositioncapable of eliciting an immune response against a tumor or against aninfectious disease pathogen such as a virus, a bacteria, a parasite or afungus. The antigen or antigens can be, for example, peptides/proteins,polysaccharides and/or lipids. The antigen or antigens be derived fromtumors, such as the various tumor antigens previously disclosed herein.Alternatively, the antigen or antigens can be derived from pathogenssuch as viruses, bacteria, parasites and/or fungi, such as the variouspathogen antigens previously disclosed herein. Additional examples ofsuitable pathogen antigens include, but are not limited to, thefollowing:

Viral antigens or antigenic determinants can be derived from, forexample: Cytomegalovirus (especially Human, such as gB or derivativesthereof); Epstein Barr virus (such as gp350); flaviviruses (e.g. YellowFever Virus, Dengue Virus, Tick-borne encephalitis virus, JapaneseEncephalitis Virus); hepatitis virus such as hepatitis B virus (forexample Hepatitis B Surface antigen such as the PreS1, PreS2 and Santigens described in EP-A-414 374; EP-A-0304 578, and EP-A-198474),hepatitis A virus, hepatitis C virus and hepatitis E virus; HIV-1, (suchas tat, nef, gp120 or gp160); human herpes viruses, such as gD orderivatives thereof or Immediate Early protein such as ICP27 from HSV1or HSV2; human papilloma viruses (for example HPV6, 11, 16, 18);Influenza virus (whole live or inactivated virus, split influenza virus,grown in eggs or MDCK cells, or Vero cells or whole flu virosomes (asdescribed by Gluck, Vaccine, 1992, 10, 915-920) or purified orrecombinant proteins thereof, such as NP, NA, HA, or M proteins);measles virus; mumps virus; parainfluenza virus; rabies virus;Respiratory Syncytial virus (such as F and G proteins); rotavirus(including live attenuated viruses); smallpox virus; Varicella ZosterVirus (such as gpI, II and IE63); and the HPV viruses responsible forcervical cancer (for example the early proteins E6 or E7 in fusion witha protein D carrier to form Protein D-E6 or E7 fusions from HPV 16, orcombinations thereof; or combinations of E6 or E7 with L2 (see forexample WO 96/26277).

Bacterial antigens or antigenic determinants can be derived from, forexample: Bacillus spp., including B. anthracis (eg botulinum toxin);Bordetella spp, including B. pertussis (for example pertactin, pertussistoxin, filamenteous hemagglutinin, adenylate cyclase, fimbriae);Borrelia spp., including B. burgdorferi (eg OspA, OspC, DbpA, DbpB), B.garinii (eg OspA, OspC, DbpA, DbpB), B. afzelii (eg OspA, OspC, DbpA,DbpB), B. andersonii (eg OspA, OspC, DbpA, DbpB), B. hermsii;Campylobacter spp, including C. jejuni (for example toxins, adhesins andinvasins) and C. coli; Chlamydia spp., including C. trachomatis (egMOMP, heparin-binding proteins), C. pneumonie (eg MOMP, heparin-bindingproteins), C. psittaci; Clostridium spp., including C. tetani (such astetanus toxin), C. botulinum (for example botulinum toxin), C. difficile(eg clostridium toxins A or B); Corynebacterium spp., including C.diphtheriae (eg diphtheria toxin); Ehrlichia spp., including E. equi andthe agent of the Human Granulocytic Ehrlichiosis; Rickettsia spp,including R. rickettsii; Enterococcus spp., including E. faecalis, E.faecium; Escherichia spp, including enterotoxic E. coli (for examplecolonization factors, heat-labile toxin or derivatives thereof, orheat-stable toxin), enterohemorragic E. coli, enteropathogenic E. coli(for example shiga toxin-like toxin); Haemophilus spp., including H.influenzae type B (eg PRP), non-typable H. influenzae, for exampleOMP26, high molecular weight adhesins, P5, P6, protein D and lipoproteinD, and fimbrin and fimbrin derived peptides (see for example U.S. Pat.No. 5,843,464); Helicobacter spp, including H. pylori (for exampleurease, catalase, vacuolating toxin); Pseudomonas spp, including P.aeruginosa; Legionella spp, including L. pneumophila; Leptospira spp.,including L. interrogans; Listeria spp., including L. monocytogenes;Moraxella spp, including M catarrhalis, also known as Branhamellacatarrhalis (for example high and low molecular weight adhesins andinvasins); Morexella Catarrhalis (including outer membrane vesiclesthereof, and OMP106 (see for example WO97/41731)); Mycobacterium spp.,including M. tuberculosis (for example ESAT6, Antigen 85A, -B or -C), M.bovis, M. leprae, M. avium, M. paratuberculosis, M. smegmatis; Neisseriaspp, including N. gonorrhea and N. meningitidis (for example capsularpolysaccharides and conjugates thereof, transferrin-binding proteins,lactoferrin binding proteins, PilC, adhesins); Neisseria mengitidis B(including outer membrane vesicles thereof, and NspA (see for example WO96/29412); Salmonella spp, including S. typhi, S. paratyphi, S.choleraesuis, S. enteritidis; Shigella spp, including S. sonnei, S.dysenteriae, S. flexnerii; Staphylococcus spp., including S. aureus, S.epidermidis; Streptococcus spp, including S. pneumonie (eg capsularpolysaccharides and conjugates thereof, PsaA, PspA, streptolysin,choline-binding proteins) and the protein antigen Pneumolysin (BiochemBiophys Acta, 1989, 67, 1007; Rubins et al., Microbial Pathogenesis, 25,337-342), and mutant detoxified derivatives thereof (see for example WO90/06951; WO 99/03884); Treponema spp., including T. pallidum (eg theouter membrane proteins), T. denticola, T. hyodysenteriae; Vibrio spp,including V. cholera (for example cholera toxin); and Yersinia spp,including Y. enterocolitica (for example a Yop protein), Y. pestis, Y.pseudotuberculosis.

Parasitic/fungal antigens or antigenic determinants can be derived from,for example: Babesia spp., including B. microti; Candida spp., includingC. albicans; Cryptococcus spp., including C. neoformans; Entamoeba spp.,including E. histolytica; Giardia spp., including; G. lamblia; Leshmaniaspp., including L. major; Plasmodium. faciparum (MSP1, AMA1, MSP3, EBA,GLURP, RAP1, RAP2, Sequestrin, PfEMP1, Pf332, LSA1, LSA3, STARP, SALSA,PfEXP1, Pfs25, Pfs28, PFS27/25, Pfs16, Pfs48/45, Pfs230 and theiranalogues in Plasmodium spp.); Pneumocystis spp., including P. carinii;Schisostoma spp., including S. mansoni; Trichomonas spp., including T.vaginalis; Toxoplasma spp., including T. gondii (for example SAG2, SAG3,Tg34); Trypanosoma spp., including T. cruzi.

It will be appreciated that in accordance with this aspect of thepresent invention antigens and antigenic determinants can be used inmany different forms. For example, antigens or antigenic determinantscan be present as isolated proteins or peptides (for example inso-called “subunit vaccines”) or, for example, as cell-associated orvirus-associated antigens or antigenic determinants (for example ineither live or killed pathogen strains). Live pathogens will preferablybe attenuated in known manner. Alternatively, antigens or antigenicdeterminants may be generated in situ in the subject by use of apolynucleotide coding for an antigen or antigenic determinant (as inso-called “DNA vaccination”), although it will be appreciated that thepolynucleotides which can be used with this approach are not limited toDNA, and may also include RNA and modified polynucleotides as discussedabove.

In one embodiment, a vaccine antigen can also be targeted, for exampleto particular cell types or to particular tissues. For example, thevaccine antigen can be targeted to Antigen Presenting Cells (APCs), forexample by use of agents such as antibodies targeted to APC-surfacereceptors such as DEC-205, for example as discussed in WO 2009/061996(Celldex Therapeutics, Inc), or the Mannose Receptor (CD206) for exampleas discussed in WO 03040169 (Medarex, Inc.).

For use in therapy, the antibodies of the invention can be administeredto a subject directly (i.e., in vivo), either alone or with othertherapies such as an immunostimulatory agent, a vaccine, chemotherapy orradiation therapy. In all cases, the antibodies, bispecifics,compositions, and immunostimulatory agents and other therapies areadministered in an effective amount to exert their desired therapeuticeffect. The term “effective amount” refers to that amount necessary orsufficient to realize a desired biologic effect. For example, aneffective amount could be that amount necessary to eliminate a tumor,cancer, or bacterial, viral or fungal infection. The effective amountfor any particular application can vary depending on such factors as thedisease or condition being treated, the particular antibody beingadministered, the size of the subject, or the severity of the disease orcondition. One of ordinary skill in the art can empirically determinethe effective amount of a particular molecule without necessitatingundue experimentation.

Preferred routes of administration for vaccines include, for example,injection (e.g., subcutaneous, intravenous, parenteral, intraperitoneal,intrathecal). The injection can be in a bolus or a continuous infusion.Other routes of administration include oral administration.

Antibodies and bispecific molecules of the invention also can becoadministered with adjuvants and other therapeutic agents. It will beappreciated that the term “coadministered” as used herein includes anyor all of simultaneous, separate, or sequential administration of theantibodies and conjugates of the present invention with adjuvants andother agents, including administration as part of a dosing regimen. Theantibodies are typically formulated in a carrier alone or in combinationwith such agents. Examples of such carriers include solutions, solvents,dispersion media, delay agents, emulsions and the like. The use of suchmedia for pharmaceutically active substances is well known in the art.Any other conventional carrier suitable for use with the molecules fallswithin the scope of the instant invention.

Suitable agents for co-administration with the antibodies, conjugates,bispecifics, and compositions include other antibodies, cytotoxinsand/or drugs, as well as adjuvants, immunostimulatory agents and/orimmunosuppressive agents. In one embodiment, the agent is achemotherapeutic agent. The antibodies, bispecifics, and compositionscan be administered in combination with radiation.

Chemotherapeutic agents suitable for coadministration with theantibodies and conjugates of the present invention in the treatment oftumors include, for example: taxol, cytochalasin B, gramicidin D,ethidium bromide, emetine, mitomycin, etoposide, tenoposide,vincristine, vinblastine, colchicin, doxorubicin, daunorubicin,dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D,1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,propranolol, and puromycin and analogs or homologs thereof. Furtheragents include, for example, antimetabolites (e.g., methotrexate,6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracildecarbazine), alkylating agents (e.g., mechlorethamine, thioepachlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cis-dichlorodiamine platinum (II) (DDP) cisplatin),anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine) and temozolomide.

Agents that delete or inhibit immunosuppressive activities, for example,by immune cells (for example regulatory T-cells, NKT cells, macrophages,myeloid-derived suppressor cells, immature or suppressive dendriticcells) or suppressive factors produced by the tumor or host cells in thelocal microenvironment of the tumor (for example, TGFbeta, indoleamine2,3 dioxygenase—IDO), may also be administered with the antibodies andconjugates of the present invention. Such agents include antibodies andsmall molecule drugs such as IDO inhibitors such as 1 methyl tryptophanor derivatives.

Suitable agents for coadministration with the antibodies and bispecificsof the present invention for treatment of such immune disorders includefor example, immunosuppressive agents such as rapamycin, cyclosporin andFK506; anti-TNFa agents such as etanercept, adalimumab and infliximab;and steroids. Examples of specific natural and synthetic steroidsinclude, for example: aldosterone, beclomethasone, betamethasone,budesonide, cloprednol, cortisone, cortivazol, deoxycortone, desonide,desoximetasone, dexamethasone, difluorocortolone, fluclorolone,flumethasone, flunisolide, fluocinolone, fluocinonide, fluocortin butyl,fluorocortisone, fluorocortolone, fluorometholone, flurandrenolone,fluticasone, halcinonide, hydrocortisone, icomethasone, meprednisone,methylprednisolone, paramethasone, prednisolone, prednisone, tixocortoland triamcinolone.

Suitable agents for coadministration with the antibodies and bispecificsof the present invention for inducement or enhancement of an immuneresponse include, for example, adjuvants and/or immunostimulatoryagents, non-limiting examples of which have been disclosed hereinbefore.A preferred immunostimulatory agent is a TLR3 agonist, such as Poly IC.

The present invention is further illustrated by the following exampleswhich should not be construed as further limiting. The contents ofSequence Listing, figures and all references, patents and publishedpatent applications cited throughout this application are expresslyincorporated herein by reference.

EXAMPLES Example 1 Generation of CD27-Specific Human MonoclonalAntibodies

Human anti-CD27 monoclonal antibodies were generated by immunizing theHC2/KCo7 strain of HuMAb® transgenic mice (“HuMAb” is a Trade Mark ofMedarex, Inc., Princeton, N.J.) with a soluble human CD27 antigen.HC2/KCo7 HuMAb mice were generated as described in U.S. Pat. Nos.5,770,429 and 5,545,806, the entire disclosures of which are herebyincorporated by reference.

Antigen and Immunization: The antigen was a soluble fusion proteincomprising a CD27 extracellular domain fused with an antibody Fc domain(recombinant human CD27-Fc chimeric protein (R&D Systems). The antigenwas mixed with Complete Freund's (Sigma) adjuvant for the firstimmunization. Thereafter, the antigen was mixed with Incomplete Freund's(Sigma). Additional mice were immunized with the soluble CD27 protein inRIBI MPL plus TDM adjuvant system (Sigma). 5-25 micrograms solublerecombinant CD27 antigen in PBS or 5×10⁶ CHO cells transfected forsurface expression of human CD27 in PBS were mixed 1:1 with theadjuvant. Mice were injected with 100 microliters of the preparedantigen into the peritoneal cavity every 14 days. Animals that developedanti-CD27 titers were given an iv injection of 10 micrograms solublerecombinant CD27 antigen three to four days prior to fusion. Mousespleens were harvested, and the isolated splenocytes used for hybridomapreparation.

Hybridoma Preparation: The P3×63Ag8.653 murine myeloma cell line (ATCCCRL 1580) was used for the fusions. RPMI 1640 (Invitrogen) containing10% FBS, and was used to culture the myeloma cells. Additional mediasupplements were added to the Hybridoma growth media, which included: 3%Origen-Hybridoma Cloning Factor (Igen), 10% FBS (Sigma), L-glutamine(Gibco) 0.1% gentamycin (Gibco), 2-mercaptoethanol (Gibco), HAT (Sigma;1.0×10⁴ M hypoxanthine, 4.0×10⁻⁷ M aminopterin, 1.6×10⁻⁵ M thymidine),or HT (Sigma; 1.0×10⁻⁴ M hypoxanthine, 1.6×10⁻⁵ M thymidine) media.

Spleen cells were mixed with the P3×63Ag8.653 myeloma cells in a 6:1ratio and pelleted by centrifugation. Polyethylene glycol was addeddropwise with careful mixing to facilitate fusion. Hybridomas wereallowed to grow out for one to two weeks until visible colonies becomeestablished. Supernatant was harvested and used for initial screeningfor human IgG via ELISA using a human kappa chain specific capture and ahuman Fc specific detection. IgG positive supernatants were then assayedfor CD27 specificity via flow cytometry or using a ELISA to detectanti-CD27.

Hybridomas producing specific human monoclonal antibodies (human mAbs;IgG) were subcloned and expanded. The human mAbs produced were thenpurified by protein A column chromatography according to standardconditions which led to the isolation of a number of antibodies ofparticular interest, which were designated as 4B7-1B3 (also referred toherein as 4B7), 3H12-1C8 (also referred to herein as 3H12), 1F5-1H5(also referred to herein as 1F5), 2C2-1A10 (also referred to herein as2C2), 2G9-1D11 (also referred to herein as 2G9), 1H8-B4 (also referredto herein as 1H8), 3H12-1E12 (also referred to herein as 3H12), 3G1-1A11(also referred to herein as 3G1) (1B10), 4A2-B11 (also referred toherein as 4A2), 3A10-G10 (also referred to herein as 3A10), 2G11-B5(also referred to herein as 2G11), 4H11-G11 (also referred to herein as4H11), 2H3-E8 (also referred to herein as 2H3), 4A7-B3 (also referred toherein as 4A7), 3H8-1B11 (also referred to herein as 3H8) and 1G5-1B9(also referred to herein as 1G5).

The hybridomas were also screened for cross-reactivity with rhesusmacaque CD27 and all were positive for binding.

Example 2 Determination of Affinity and Rate Constants of Human mAbs bySurface Plasmon Resonance (SPR)

Binding affinity and binding kinetics of various human anti-CD27antibodies from Example 1 were examined by Biacore™ surface plasmonresonance (SPR) analysis using a Biacore™ 2000 SPR instrument (BiacoreAB, Uppsala, Sweden) according to the manufacturer's guidelines.

Purified recombinant human CD27/TNFRSF7/Fc chimera (R&D Systems CatalogNo. 382-CD) protein was covalently linked to a Biacore™ CMS sensor chip(carboxymethylated dextran covalently attached to a gold surface;Biacore Product No. BR-1000-14) using standard amine coupling chemistrywith an Amine Coupling Kit provided by Biacore according to themanufacturer's guidelines (BIAcore Product No. BR-1000-50, comprisingcoupling reagents N-hydroxysuccinimide (NHS) and1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)). Lowlevels of ligand were immobilised to limit any effects of mass transportof analyte on kinetic parameters, such that the R_(MAX) observed was inthe order of 100-400 RU.

Binding was measured by flowing the antibodies over the sensor chip inHBS-EP buffer (HBS-EP buffer, Biacore Product No. BR-1001-88:4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) 0.01M, sodiumchloride 0.15M ethylenediaminetetraacetic acid (EDTA) 3 mM, SurfactantP20 0.005%), at concentrations ranging from 1.56 to 50 nM and at a flowrate of 30 μl/minute for 180 seconds. The antigen-antibody associationand dissociation kinetics were followed for either 480 seconds or 1200seconds for antibodies with slower dissociation rates.

Corresponding controls were conducted in each case using a blankflowcell with no protein immobilized for “background” subtraction.Sequential injections of 10 mM HCl for 10 seconds at 50 μl/min followedby 10 mM Glycine pH 2.0 for 30 seconds at 50 μl/min was used as theregeneration conditions throughout the study.

Biacore's BIAevaluation software version 3.2 (Biacore AB, Uppsala,Sweden) was used in each case to derive kinetic parameters from theconcentration series of analyte diluted in HBS-EP running buffer. Theassociation and dissociation curves were fitted to a 1:1 Langmuirbinding model using Biacore™ BIAevaluation software (Biacore AB)according to the manufacturer's guidelines.

The affinity and kinetic parameters (with background subtracted) asdetermined are shown in FIG. 1.

For each antibody, the figures shown are the mean of two separate seriesof experiments, using separately prepared flow cells in each case,(where ka=rate constant of association, kd=rate constant ofdissociation, K_(D)=dissociation equilibrium constant (measure ofaffinity), K_(A)=association equilibrium constant, Rmax=maximum SPRresponse signal).

Example 3 ELISA Assay to Determine Human mAb Binding Characteristics onCD27

Microtiter plates were coated with soluble or recombinant human ormacaque CD27 in PBS, and then blocked with 5% bovine serum albumin inPBS. Protein A purified human mAbs and an isotype control were added atsaturating concentrations and incubated at 37° C. The plates were washedwith PBS/Tween and then incubated with a goat-anti-human IgG Fc-specificpolyclonal reagent conjugated to alkaline phosphatase at 37° C. Afterwashing, the plates were developed with pNPP substrate (1 mg/ml), andanalyzed at OD 405-650 using a microtiter plate reader. Representativesbinding curves are shown in FIG. 2. The results were also used toestimate the 50% saturating concentration (C value in 4-parameter fitcurve) as shown in Table 1 below.

To establish that cynomolgus macques are a relevant model for testinganti-CD27 mAbs, various concentrations of purified macaque CD27 or humanCD27 were captured to ELISA plates with anti-Flag antibody, followed byincubation with anti-human CD27 mAb. A goat anti-human IgG Fc-HRPantibody and substrate Super Blue TMB were used for detection. Resultsare shown in Table 1, which indicate similar binding to CD27 from macqueand human. Representative binding curves for antibody 1F5 are also shownin FIG. 3.

TABLE 1 Characterization of selected anti-CD27 mAb Half-max Half-maxHalf-max binding to binding to binding to human human monkey mAbCD27(M)** CD27(μg/ml)** CD27(μg/ml)** 1G5 4.9E−10 0.074 0.065 1H84.3E−10 0.064 0.105 3H12 5.7E−10 0.085 0.123 3H8 4.6E−10 0.069 0.065 2G94.3E−10 0.064 0.069 1F5 3.9E−10 0.059 0.12 3A10 6.3E−10 0.094 no binding2C2 3.0E−10 0.045 0.034 **estimated by binding to CD27 coated plates inan ELISA format

In a further experiment, to establish similar distribution of 1F5binding to peripheral blood cells, PBMCs were isolated from whole bloodof 3 humans and 3 cynomolgus macaques. The cells were stained with 1F5mAb together with markers to delineate the major T cell and B cellpopulations that express CD27. The following table (Table 2) summarizesthe mean±standard error of results for human and macaque cells withrespect to the percent of cells expressing CD27 and the intensity ofexpression (MFI). These data establish a similar distribution of 1F5binding to peripheral blood cells from human and monkey.

TABLE 2 CD4+ T cells CD8+ T cells B cells (CD20+) NK cells Analysishuman monkey human monkey human monkey human monkey % CD27+^(b) 84 ± 581 ± 1  70 ± 12 90 ± 1 37 ± 4  15 ± 1  11 ± 4  88 ± 6 MFI^(c) 1517 ± 123416 ± 14 1415 ± 153 519 ± 11 893 ± 101 491 ± 113 667 ± 28 1050 ± 42

Example 4 4A: Blocking of sCD70 Binding by ELISA

The effect of the human mAbs from Example 1 on the binding of solubleCD70 (sCD70) to CD27 protein was measured by ELISA. A microtiter platewas coated with 1 μg/ml soluble recombinant human CD27/Fc chimera fromR&D Systems at 1 μg/mL., then blocked with 5% PBA. The anti-CD27antibodies ([final]=25 μg/mL) were premixed with soluble humanrecombinant CD70-biotin from US Biologicals ([final]=0.5 μg/mL) andadded to the plate. CD27-captured rCD70 was detected withstreptavidin-HRP and substrate Super Blue TMB. The results (shown as %blocking) are shown in FIG. 4 with controls as indicated. These resultsshow that several of the antibodies (including 1F5, 1H8, 3H12 and 1A4)had the property of blocking or at least significantly inhibiting thebinding of sCD70.

4B: CD27 mAb Binds to CD27 on Human Lymphoblastoid Cell Lines and BlocksLigand (CD70) Binding

Binding of anti-human CD27 mAb 1F5 to human lymphoblastoid cell lines,and blocking of sCD70 binding, was analysed by flow cytometry using aBecton Dickinson FACSCanto II flow cytometer. The results are shown inFIG. 5, and show that 1F5 effectively binds to a variety of cell linesand competitively inhibits binding of sCD70.

Example 5 Binding of Human mAbs to Cells Expressing Human CD27

The ability of anti-CD27 human mAbs to bind to CD27 on cells expressinghuman CD27 on their surface was investigated by flow cytometry asfollows.

Antibodies were tested for binding to human cell lines expressing humanD27 on their surface. Protein A purified human mAbs 2C2, 3H8 and 1F5were incubated with Jurkat, Raji, Ramos and Daudi cells expressing humanCD27, as well as control cells at 4° C. All antibodies were used atsaturating concentrations. After 1 hour, the cells were washed with PBScontaining 0.1% BSA and 0.05% NaN₃ (PBA) and the bound antibodies weredetected by incubating the cells with a PE labeled goat anti-human IgGFc-specific probe, at 4° C. The excess probe was washed from the cellswith PBA and the cell associated fluorescence was determined by analysisusing a LSR™ instrument (BD Biosciences, NJ, USA) according to themanufacturer's directions.

As shown in FIGS. 6 and 7, the human mAbs demonstrated high levelbinding to cells expressing human CD27. These data demonstrate thatthese antibodies bind efficiently and specifically to human CD27expressed on live cells compared to the control cells.

Example 6 Cross-Blocking/Competition of Human mAbs Determined by ELISA

A microtiter plate was coated with a recombinant human CD27-Fc chimericfusion protein, then blocked with 5% BSA in PBS. Unconjugated human mAbs(20 μg/mL) were mixed with horseradish peroxidase labeled secondaryantibodies (0.5 μg/mL), then added to the plate and incubated at 37° C.The plates were washed with PBS/Tween and developed with TMB substrateand analyzed at OD 450 using a microtiter plate reader. The results,shown in FIGS. 8 to 10, indicate that a first set of the human mAbs(comprising mAbs 1F5, 1H8 and 3H12) cross-competed with each other (seeFIG. 8), that a further set of the human mAbs (comprising mAbs 2C2, 3H8,1G5, and 2G9) also cross-competed with each other (see FIG. 9), and thathuman mAb 3A10 binds a unique epitope but may bind at a site distinctfrom but possibly close to the binding sites of mAbs 1F5, 1H8, and 3H12since these antibodies were able to partially cross-block the binding of3A10 to CD27 (see FIG. 10).

Example 7 Complement Dependent Cellular Cytotoxicity (CDCC or CDC)

Target cells (Lymphoma Raji cells) were cultured (in AIM-V medium) for1-2 hours at 37° C., 5% CO₂ in the presence of anti-CD27 antibodies andrabbit complement (final dilution of 1:15) in a final volume of 150 μl.Appropriate controls with a leak signal (targets only) and MAX signal(targets with Lysol™ detergent at 12% for a final concentration of 4%)were included as well. Cells were adjusted to 1×10⁶/ml and 50 μl wereadded to each well (to give 50,000 cells/well). Wells were thenresuspended and 100 μl of the cell suspension was transferred to anopaque, white plate. To each of these wells, 100 uL of Promega CellTiterGlo reagent was added and the plate was mixed for 2 minutes at roomtemperature. The plate was allowed to incubate for 10 minutes tostabilize the luminescent signal. Luminescence was recorded on a PerkinElmer Victor X4 plate reader. Cytotoxicity was determined with thefollowing formula: (100−((sample−MAX)/(leak−MAX)))×100. Results (shownas % lysis) are shown in FIG. 11 from which it can be seen that a numberof the anti-CD27 antibodies displayed significant CDCC activity.

In a further experiment, target cells (Ramos cells) were washed andloaded with Calcein AM (Molecular Probes). Loaded cells were then washedagain and resuspended to 1×10⁶/ml in culture media (RPMI+10% FBS).Target cells were cultured for 2 hours at 37° C., 5% CO₂ in the presenceof anti-CD27 antibodies and rabbit complement (final dilution of 1:15)in a final volume of 150 μl. Appropriate controls with a leak signal(targets only) and MAX signal (targets with Triton X-100 at 20% for afinal concentration of 2%) were included as well. Following theincubation, 75 μl of supernatant from the wells were transferred into anopaque, black plate. Fluorescence (Ex 485; Em 535) was recorded on aPerkin Elmer Victor X4 plate reader. Specific cytotoxicity wasdetermined using the following formula: (experimental−spontaneouslysis)/(maximum lysis−spontaneous lysis)×100. The results, shown in FIG.12, indicate that anti-CD27 mAb (1F5) showed at least 10% CDC activityin Ramos cells at an antibody concentration of 3 μg/ml.

Example 8 Antibody Dependent Cell-Mediated Cytotoxicity (ADCC)

Targets cells (Lymphoma Raji cells) were washed and loaded with BATDAreagent (Perkin Elmer). Loaded cells were then washed again andresuspended to 2×10⁵/ml in culture media (RPMI+10% FBS). Effector cellswere prepared and adjusted to appropriate concentrations in culturemedia to yield desired effector:target ratios (100:1-50:1). In a roundbottom plate, target cells, effector cells and antibody were added in afinal volume of 150 μl. Appropriate controls were used, including a leaksignal (targets only), a spontaneous lysis signal (targets+effectors),and a maximum lysis signal (targets+Lysol™ detergent at 12% for a finalconcentration of 4%). Cells were pelleted in the plate and incubated for2 hours at 37° C., 5% CO₂. Following the incubation, 20 μl ofsupernatant from the wells were transferred into an opaque, white plate.To each of these wells, 200 μl of Europium solution (Perkin Elmer) wasadded and the plate was mixed for 15 minutes. Time-resolved fluorescencewas recorded on a Perkin Elmer Victor X4 plate reader. Specificcytotoxicity was determined using the following formula:(experimental−spontaneous lysis)/(maximum lysis−spontaneous lysis)×100.Results (shown as % lysis) are shown in FIG. 13 from which it can beseen that a number of the anti-CD27 antibodies displayed significantADCC activity.

In a further experiment, target cells (Lymphoma Ramos and Daudi cells)were washed and loaded with Calcein AM (Molecular Probes) Loaded cellswere then washed again and resuspended to 1×10⁵/ml in culture media(RPMI+10% FBS). Effector cells were prepared and adjusted to appropriateconcentrations in culture media to yield desired effector:target ratios(75:1). In a round bottom plate, target cells, effector cells andantibody were added in a final volume of 150 μl. Appropriate controlswere used, including a leak signal (targets only), a spontaneous lysissignal (targets+effectors), and a maximum lysis signal (targets+TritonX-100 at 20% for a final concentration of 2%). Cells were pelleted inthe plate and incubated for 4 hours at 37° C., 5% CO₂. Following theincubation, 75 μl of supernatant from the wells were transferred into anopaque, black plate. Fluorescence (Ex 485; Em 535) was recorded on aPerkin Elmer Victor X4 plate reader. Specific cytotoxicity wasdetermined using the following formula: (experimental−spontaneouslysis)/(maximum lysis−spontaneous lysis)×100. The results, shown in FIG.14 indicate that anti-CD27 mAb (1F5) showed at least 10% ADCC activity(measured as specific cytotoxicity) in Daudi and Ramos cells at anantibody concentration of 3 μg/ml and ratio of effector:target cells of75:1.

Example 9 Antibody Sequencing

As described above in Example 1, human mAbs from hybridomas producingspecific human mAbs IgG were purified by protein A column chromatographywhich led to the isolation of a panel of antibodies (human mAbs) ofparticular interest. The V_(H) and V_(L) coding regions of human mAbs4B7, 3H12, 1F5, 2C2, 2G9, 1H8, 3H12, 3G1 (1B10) 4A2, 3A10, 2G11, 4H11,2H3, 4A7, 3H8 and 1G5 were identified using RNA from the correspondinghybridomas. RNA was reverse transcribed to cDNA, the V coding regionswere amplified by PCR and the PCR product was sequenced. The followingare the nucleic and amino acid sequences of the V_(H) and V_(L) regionsof the human mAbs (in the case of the amino acid sequences, theComplementarity Determining Regions (CDRs) are underlined).

3H8 VH (VH 3-7; D7-27; JH2)

V_(H) nucleic acid sequence (SEQ ID NO: 5)

atggagttggggctgagctgggttttccttgttgctattttagaaggtgtccagtgtgaggtgcagctggtggagtctgggggaggcttggtccagcctggggggtccctgagactctcctgtgcagcctctggattcacctttagtagttattggatggcctgggtccgccaggctccagggaaagggctggagtggctgggcaatataaagcaagatggaagtgagaaatactatgtggactctgtgaagggccgattcaccatctccagagacaacgccaagaactcactgtatctacaaatgaacagcctgagagccgaggacacggctgtgtattactgtgtgagggaactggggatggactggtacttcgatctctggggccgtgg caccctggtcactgtctcctcaV_(H) amino acid sequence (SEQ ID NO: 6) (including signal peptide inunderlined italics):

MELGLSWVFLVAILEGVQC EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMAWVRQAPGKGLEWLGNIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCVRELGMDWYFDLWGRGTLVTVSSV_(H) “mature” amino acid sequence (SEQ ID NO: 7) excluding signalpeptide:

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMAWVRQAPGKGLEWLGNIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCVR ELGMDWYFDLWGRGTLVTVSSV_(H )CDR1 (SEQ ID NO: 8): GFTFSSYW V_(H )CDR2 (SEQ ID NO: 9): IKQDGSEKV_(H )CDR3V (SEQ ID NO: 10): VRELGMDWYFDL3H8 VK #2 (VK 3-11; JK1)V_(L) nucleic acid sequence (SEQ ID NO: 11)

AtggaagccccagctcagcttctcttcctcctgctactctggctcccagataccaccggagaaattgtgttgacacagtctccagccaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttgacagctacttagcctggtaccaacagaaacctggccaggctcccaggctcctcatctatgatgcatccaacagggccactggcatcccagccaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcaacctagagcctgaagattttgcagtttattactgtcagcagcgtagcaactggcctccgacgttcggccaagggaccaaggtggaaatcaaaV_(L) amino acid sequence (SEQ ID NO: 12) (including signal peptide inunderlined italics):

MEAPAQLLFLLLLWLPDTTG EIVLTQSPATLSLSPGERATLSCRASQSVDSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISNLEPEDFAVYYCQQRSNWPPTFGQGTKVEIKV_(L) amino acid sequence (SEQ ID NO: 13) excluding signal peptide:

EIVLTQSPATLSLSPGERATLSCRASQSVDSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISNLEPEDFAVYYCQQRSNWPPTF GQGTKVEIKV_(L )CDR1 (SEQ ID NO: 14): QSVDSY V_(L )CDR2 (SEQ ID NO: 15): DASV_(L )CDR3 (SEQ ID NO: 16): QQRSNWPPT3H8 VK #3 (VK 3-11; JK1)A further light chain was also found to be active as follows (thoughonly the above light chain (3H8-1B11 VK #2) was used in the aboveExamples):V_(L) nucleic acid sequence (SEQ ID NO: 17)

AtggaagccccagctcagcttctcttcctcctgctactctggctcccagataccaccggagaaattgtgttgacacagtctccagccaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttagcagctacttagcctggtaccaacagaaacctggccaggctcccaggctcctcatctatgatgcatccagcagggccactggcatcccagacaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagactggagcctgaagattttgcagtgtattactgtcagcagcgtagcaactggcctccgacgttcggccaagggaccaaggtggaaatcaaaV_(L) amino acid sequence (SEQ ID NO: 18) (including signal peptide inunderlined italics):

MEAPAQLLFLLLLWLPDTTG EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQRSNWPPTFGQGTKVEIKV_(L) amino acid sequence (SEQ ID NO: 19) excluding signal peptide:

QSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQRSNPPTFGQG TKVEIKV_(L )CDR1 (SEQ ID NO: 20): QSVSSY V_(L )CDR2 (SEQ ID NO: 21): DASV_(L )CDR3 (SEQ ID NO: 22): QQRSNWPPT2C2 VH (VH 3-33; D1-7; JH4)V_(H) nucleic acid sequence (SEQ ID NO: 23)

Atggagtttgggctgagctgggttttcctcgttgctcttttaagaggtgtccagtgtcaggtgcaactggtggagtctgggggaggcgtggtccagcctgggaggtccctgcgactctcctgtgcagcgtctggattcaccttcagtagctatgacatacactgggtccgccaggctccaggcaaggggctggagtgggtggcagttatatggaatgatggaagtaataaatactatgcagactccgtgaagggccgattcaccatctccagagacaattccacgaactcgctgtttctgcaaatgaacagcctgagagccgaggacacggctgtgtattattgtgtgggaggaactgctgaccttgaacactgggaccagggaaccctggtcaccgtct cctcaV_(H) amino acid sequence (SEQ ID NO: 24) (including signal peptide inunderlined italics):

MEFGLSWVFLVALLRGVQC QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYDIHWVRQAPGKGLEWVAVIWNDGSNKYYADSVKGRFTISRDNSTNSLFLQMNSLRAEDTAVYYCVGGTADLEHWDQGTLVTVSSV_(H) amino acid sequence (SEQ ID NO: 25) excluding signal peptide:

QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYDIHWVRQAPGKGLEWVAVIWNDGSNKYYADSVKGRFTISRDNSTNSLFLQMNSLRAEDTAVYYCVGGT ADLEHWDQGTLVTVSSV_(H) CDR1 (SEQ ID NO: 26): GFTFSSYD V_(H) CDR2 (SEQ ID NO: 27):IWNDGSNK V_(H) CDR3 (SEQ ID NO: 28): VGGTADLEHWDQ2C2 VK (VK 1D-16; JK4)V_(L) nucleic acid sequence (SEQ ID NO: 29)

AtgagggtcctcgctcagctcctggggctcctgctgctctgtttcccaggtgccagatgtgacatccagatgacccagtctccatcctcactgtctgcatctgtaggagacagagtcaccatcacttgtcgggcgagtcagggtattagcagctggttagcctggtatcagcagaaaccagagaaagcccctaagtccctgatctatgctgcatccagtttgcaaagtggggtcccatcaaggttcagcggcagtggatctgggacagatttcactctcaccatcagcagcctgcagcctgaagattttgcaacttattactgccaacagtataatagttaccctctcactttcggcggagggaccaaggtggagatcaaaV_(L) amino acid sequence (SEQ ID NO: 30) (including signal peptide inunderlined italics):

MRVLAQLLGLLLLCFPGARC DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPLTFGGGTKVEIKV_(L) amino acid sequence (SEQ ID NO: 31) excluding signal peptide:

DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPLTFGG GTKVEIK V_(L) CDR1(SEQ ID NO: 32): QGISSW V_(L) CDR2 (SEQ ID NO: 33): AAS V_(L) CDR3 (SEQID NO: 34): QQYNSYPLT1F5 VH (VH 3-33; D7-27; JH4)V_(H) nucleic acid sequence (SEQ ID NO: 35)

Atggagtttgggctgagctgggttttcctcgttgctcttttaagaggtgtccagtgtcaggtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccctgagactctcctgtgcagcgtctggattcaccttcagtagttatgacatgcactgggtccgccaggctccaggcaaggggctggagtgggtggcagttatatggtatgatggaagtaataaatactatgcagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctgtatctccaaatgaacagcctgagagccgaggacacggctgtgtattactgtgcgagaggtagtggtaactggggtttctttgactactggggccagggaaccctgg tcaccgtctcctcaV_(H) amino acid sequence (SEQ ID NO: 36) (including signal peptide inunderlined italics):

MEFGLSWVFLVALLRGVQC QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYDMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGSGNWGFFDYWGQGTLVTVSSV_(H) amino acid sequence (SEQ ID NO: 37) excluding signal peptide:

QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYDMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGS GNWGFFDYWGQGTLVTVSSV_(H) CDR1 (SEQ ID NO: 38): GFTFSSYD V_(H) CDR2 (SEQ ID NO: 39):IWYDGSNK V_(H) CDR3 (SEQ ID NO: 40): ARGSGNWGFFDY1F5 VK #2 (VK 1D-16; JK1)V_(L) nucleic acid sequence (SEQ ID NO: 41)

AtgagggtcctcgctcagctcctggggctcctgctgctctgtttcccaggtgccagatgtgacatccagatgacccagtctccatcctcactgtctgcatctgtaggagacagagtcaccatcacttgtcgggcgagtcagggtattagcaggtggttagcctggtatcagcagaaaccagagaaagcccctaagtccctgatctatgctgcatccagtttgcaaagtggggtcccatcaaggttcagcggcagtggatctgggacagatttcactctcaccatcagcagcctgcagcctgaagattttgcaacttattactgccaacagtataatacttaccctcggacgttcggccaagggaccaaggtggaaatcaaaV_(L) amino acid sequence (SEQ ID NO: 42) (including signal peptide inunderlined italics):

MRVLAQLLGLLLLCFPGARC DIQMTQSPSSLSASVGDRVTITCRASQGISRWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNTYPRTFGQGTKVEIKV_(L) amino acid sequence (SEQ ID NO: 43) excluding signal peptide:

DIQMTQSPSSLSASVGDRVTITCRASQGISRWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNTYPRTFGQ GTKVEIK V_(L) CDR1(SEQ ID NO: 44): QGISRW V_(L) CDR2 (SEQ ID NO: 45): AAS V_(L) CDR3 (SEQID NO: 46): QQYNTYPRT1H8 VH (VH 3-33; D7-27; JH4)V_(H) nucleic acid sequence (SEQ ID NO: 47)

Atggagtttgggctgagctgggttttcctcgttgctcttttaagaggtgtccagtgtcaggtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccctgagactctcctgtgcagcgtctggattcaccttcaatatctatgacatgcactgggtccgccaggctccaggcaaggggctggagtgggtggcagttatatggtatgatggaagtaatcaatactatgcagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacattttgagagccgaggacacggctgtgtattactgtgcgagaggtactcactgggggtactttgactactggggccagggaaccctggtca ccgtctcctcaV_(H) amino acid sequence (SEQ ID NO: 48) (including signal peptide inunderlined italics):

MEFGLSWVFLVALLRGVQC QVQLVESGGGVVQPGRSLRLSCAASGFTFNIYDMHWVRQAPGKGLEWVAVIWYDGSNQYYADSVKGRFTISRDNSKNTLYLQMNILRAEDTAVYYCARGTHWGYFDYWGQGTLVTVSSV_(H) amino acid sequence (SEQ ID NO: 49) excluding signal peptide:

QVQLVESGGGVVQPGRSLRLSCAASGFTFNIYDMHWVRQAPGKGLEWVAVIWYDGSNQYYADSVKGRFTISRDNSKNTLYLQMNILRAEDTAVYYCARGT HWGYFDYWGQGTLVTVSSV_(H) CDR1 (SEQ ID NO: 50): GFTFNIYD V_(H) CDR2 (SEQ ID NO: 51):IWYDGSNQ V_(H) CDR3 (SEQ ID NO: 52): ARGTHWGYFDY1H8 VK (VK 1D-16; JK1)V_(L) nucleic acid sequence (SEQ ID NO: 53)

AtgagggtcctcgctcagctcctggggctcctgctgctctgtttcccaggtgccagatgtgacatccagatgacccagtctccatcctcactgtctgcatctgtaggagacagagtcaccatcacttgtcgggcgagtcagggtattagcagctggttagcctggtatcagcagaaaccagagaaagcccctaagtccctgatctatgctgcatccaatttgcaaagtggggtcccatcaaggttcagcggcagtggatctgggacagatttcactctcaccatcagcagcctgcagcctgaagattttgcaacttattactgccaacagtataatagttaccctcggacgttcggccaagggaccaaggtggaaatcaaaV_(L) amino acid sequence (SEQ ID NO: 54) (including signal peptide inunderlined italics):

MRVLAQLLGLLLLCFPGARC DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPRTFGQGTKVEIKV_(L) amino acid sequence (SEQ ID NO: 55) excluding signal peptide:

DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPRTF GQGTKVEIKV_(L )CDR1 (SEQ ID NO: 56): QGISSW V_(L )CDR2 (SEQ ID NO: 57): AASV_(L )CDR3 (SEQ ID NO: 58): QQYNSYPRT1G5 VH (VH 3-33; D6-19; JH2)V_(H) nucleic acid sequence (SEQ ID NO: 59)

AtggagtttgggctgagctgggttttcctcgttgctcttttaagaggtgtccagtgtcaggtgcaactggtggagtctgggggaggcgtggtccagcctgggaggtccctgagactctcctgtgcagcgtctggattcagcttcagtagctatggcatgcactgggtccgccaggctccaggcaagggactggagtgggtggcacttctatggtatgatggtagccataaagactttgcagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctagatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtgcgagagagggtttagcagtacctggtcactggtacttcgatctctggggccgtggcaccctggtcactgtctcctcaV_(H) amino acid sequence (SEQ ID NO: 60) (including signal peptide inunderlined italics):

MEFGLSWVFLVALLRGVQC QVQLVESGGGVVQPGRSLRLSCAASGFSFSSYGMHWVRQAPGKGLEWVALLWYDGSHKDFADSVKGRFTISRDNSKNTLDLQMNSLRAEDTAVYYCAREGLAVPGHWYFDLWGRGTLVTVSSV_(H) amino acid sequence (SEQ ID NO: 61) excluding signal peptide:

QVQLVESGGGVVQPGRSLRLSCAASGFSFSSYGMHWVRQAPGKGLEWVALLWYDGSHKDFADSVKGRFTISRDNSKNTLDLQMNSLRAEDTAVYYCAREGLAVPGHWYFDLWGRGTLVTVSS V_(H )CDR1 (SEQ ID NO: 62): GFSFSSYGV_(H )CDR2 (SEQ ID NO: 63): LWYDGSHK V_(H )CDR3 (SEQ ID NO: 64):AREGLAVPGHWYFDL1G5 VK (VK 1-13; JK1)V_(L) nucleic acid sequence (SEQ ID NO: 65)

AtgagggtccccgctcagctcctggggcttctgctgctctggctcccaggtgccagatgtgccatccagttgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagtcagggcattagcagtgctttagcctggtatcagcagaaaccagggaaagctcctaagctcctgatctatgatgcctccagtttggaaagtggggtcccatcaaggttcagcggcagtggatctgggacagatttcactctcaccatcagcagcctgcagcctgaagattttgcaacttattactgtcaacagtttaatacttaccctcggacgttcggccaagggaccaaggtggaaatcaaaV_(L) amino acid sequence (SEQ ID NO: 66) (including signal peptide inunderlined italics):

MRVPAQLLGLLLLWLPGARC AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNTYPRTFGQGTKVEIKV_(L) amino acid sequence (SEQ ID NO: 67) excluding signal peptide:

AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNTYPRTF GQGTKVEIKV_(L )CDR1 (SEQ ID NO: 68): QGISSA V_(L )CDR2 (SEQ ID NO: 69): DASV_(L )CDR3 (SEQ ID NO: 70): QQFNTYPRT2G9 VH (VH 3-33; D1-7; JH4)V_(H) nucleic acid sequence (SEQ ID NO: 71)

Atggagtttgggctgagctgggttttcctcgttgctcttttaagaggtgtccagtgtcaggtgcagttggtggagtctgggggaggcgtggtccagcctgggaggtccctgcgactctcctgtgcagcgtctggattcaccctcagtagccatgacatacactgggtccgccaggctccaggcaaggggctggagtgggtggcagttatatggaatgatggaagtaataaatactatgcagactccgtgaagggccgattcaccatctccagagacaattccacgaactcgctgtttctgcaaatgaacagcctgagagccgaggacacggctgtgtattattgtgtgagaggaactgctgaccttgaacactgggaccagggaaccctggt caccgtctcctcaV_(H) amino acid sequence (SEQ ID NO: 72) (including signal peptide inunderlined italics):

MEFGLSWVFLVALLRGVQC QVQLVESGGGVVQPGRSLRLSCAASGFTLSSHDIHWVRQAPGKGLEWVAVIWNDGSNKYYADSVKGRFTISRDNSTNSLFLQMNSLRAEDTAVYYCVRGTADLEHWDQGTLVTVSSV_(H) amino acid sequence (SEQ ID NO: 73) excluding signal peptide:

QVQLVESGGGVVQPGRSLRLSCAASGFTLSSHDIHWVRQAPGKGLEWVAVIWNDGSNKYYADSVKGRFTISRDNSTNSLFLQMNSLRAEDTAVYYCVR GTADLEHWDQGTLVTVSSV_(H )CDR1 (SEQ ID NO: 74): GFTLSSHD V_(H )CDR2 (SEQ ID NO: 75):IWNDGSNK V_(H )CDR3 (SEQ ID NO: 76): VRGTADLEHWDQ2G9 VK (VK 1D-16; JK4)V_(L) nucleic acid sequence (SEQ ID NO: 77)

AtgagggtcctcgctcagctcctggggctcctgctgctctgtttcccaggtgccagatgtgacatccagatgacccagtctccatcctcactgtctgcatctgtaggagacagagtcaccatcacttgtcgggcgagtcagggtattagcagctggttagcctggtatcagcagaaaccagagaaagcccctaagtccctgatctatgctgcatccagtttgcaaagtggggtcccatcaaggttcagcggcagtggatctgggacagatttcactctcaccatcagcagcctgcagcctgaagattttgcaacttattactgccaacagtataatagttaccctctcactttcggcggagggaccaaggtggagatcaaaV_(L) amino acid sequence (SEQ ID NO: 78) (including signal peptide inunderlined italics):

MRVLAQLLGLLLLCFPGARC DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPLTFGGGTKVEIKV_(L) amino acid sequence (SEQ ID NO: 79) excluding signal peptide:

DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPLTF GGGTKVEIKV_(L )CDR1 (SEQ ID NO: 80): QGISSW V_(L )CDR2 (SEQ ID NO: 81): AASV_(L )CDR3 (SEQ ID NO: 82): QQYNSYPLT3A10 VH (VH 3-33; D3-10; JH3)V_(H) nucleic acid sequence (SEQ ID NO: 83)

AtggagtttgggctgagctgggttttcctcgttgctcttttaagaggtgtccagtgtcaggtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccctgagactctcctgtgcagcgtctggattcaccttcagtcattatggcatgcactgggtccgccaggctccaggcaaggggccggagtgggtggcaattatatggtatgatggaagtaataaatactatgcagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctggatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtgcgagagatggatggactactatggttcggggacttaatgtttttgatatctggggccaagggacaatggtcaccgtctcttcaV_(H) amino acid sequence (SEQ ID NO: 84) (including signal peptide inunderlined italics):

MEFGLSWVFLVALLRGVQC QVQLVESGGGVVQPGRSLRLSCAASGFTFSHYGMHWVRQAPGKGPEWVAIIWYDGSNKYYADSVKGRFTISRDNSKNTLDLQMNSLRAEDTAVYYCARDGWTTMVRGLNVFDIWGQGTMVTVSSV_(H) amino acid sequence (SEQ ID NO: 85) excluding signal peptide:

QVQLVESGGGVVQPGRSLRLSCAASGFTFSHYGMHWVRQAPGKGPEWVAIIWYDGSNKYYADSVKGRFTISRDNSKNTLDLQMNSLRAEDTAVYYCARDGWTTMVRGLNVFDIWGQGTMVTVSS V_(H) CDR1 (SEQ ID NO: 86): GFTFSHYG V_(H)CDR2 (SEQ ID NO: 87): IWYDGSNK V_(H) CDR3 (SEQ ID NO: 88):ARDGWTTMVRGLNVFDI3A10 VK #1 (VK 1D-16; JK5)V_(L) nucleic acid sequence (SEQ ID NO: 89)

AtgagggtcctcgctcagctcctggggctcctgctgctctgtttcccaggtgccagatgtgacatccagatgacccagtctccatcctcactgtctgcatctgtaggagacagagtcaccatcacttgtcgggcgagtcaggatattagcagctggttagcctggtatcagcagaaaccagagaaagcccctaagtccctgatctatgctgcatccagtttgcaaagtggggtcccatcaaggttcagcggcagtggatctgggacagatttcactctcaccatcagcagcctgcagcctgaagattttgcaacttattactgccaacagtataatagttaccctcccaccttcggccaagggacacgactggagattaaaV_(L) amino acid sequence (SEQ ID NO: 90) (including signal peptide inunderlined italics):

MRVLAQLLGLLLLCFPGARC DIQMTQSPSSLSASVGDRVTITCRASQDISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPPTFGQGTRLEIKV_(L) amino acid sequence (SEQ ID NO: 91) excluding signal peptide:

DIQMTQSPSSLSASVGDRVTITCRASQDISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPPTF GQGTRLEIK V_(L) CDR1(SEQ ID NO: 92): QDISSW V_(L) CDR2 (SEQ ID NO: 93): AAS V_(L) CDR3 (SEQID NO: 94): QQYNSYPPT3A10 VK #4 (VK 1-13; JK5)A further light chain was also found to be active as follows (thoughonly the above light chain (3H8-1B11 VK #1) was used in the aboveExamples):V_(L) nucleic acid sequence (SEQ ID NO: 95)

AtgagggtcctcgctcagctcctggggcttctgctgctctggctcccaggtgccagatgtgccatccagttgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagtcagggcattagcagtgctttagcctggtatcagcagaaaccagagaaagcccctaagtccctgatctatgctgcatccagtttgcaaagtggggtcccatcaaggttcagcggcagtggatctgggacagatttcactctcaccatcagcagcctgcagcctgaagattttgcaacttattactgccaacagtataatagttaccctcccaccttcggccaagggacacgactggagattaaaV_(L) amino acid sequence (SEQ ID NO: 96) (including signal peptide inunderlined italics):

MRVLAQLLGLLLLWLPGARC AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPPTFGQGTRLEIKV_(L) amino acid sequence (SEQ ID NO: 97) excluding signal peptide:

AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPPTF GQGTRLEIK V_(L) CDR1(SEQ ID NO: 98): QGISSA V_(L) CDR2 (SEQ ID NO: 99): AAS V_(L) CDR3 (SEQID NO: 100): QQYNSYPPT3H12 VH (VH 3-33; D7-27; JH4)V_(H) nucleic acid sequence (SEQ ID NO: 101)

atggagtttgggctgagctgggttttcctcgttgctcttttaagaggtgtccagtgtcaggtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccctgagactctcctgtgcaacgtctggattcaccttcagtagctatgacatgcactgggtccgccaggctccaggcaaggggctggagtgggtggcagttatttggtatgatggaagtaataaatactatgcagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctgtatctccaaatgaacagcctgggagacgaggacacggctgtgtattactgtgcgagaggtagtggtaactggggtttctttgactactggggccaggg aaccctggtcaccgtctcctcaV_(H) amino acid sequence (SEQ ID NO: 102) (including signal peptide inunderlined italics):

MEFGLSWVFLVALLRGVQC QVQLVESGGGVVQPGRSLRLSCATSGFTFSSYDMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLGDEDTAVYYCARGSGNWGFFDYWGQGTLVTVSSV_(H) amino acid sequence (SEQ ID NO: 103) excluding signal peptide:

QVQLVESGGGVVQPGRSLRLSCATSGFTFSSYDMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLGDEDTAVYYCAR GSGNWGFFDYWGQGTLVTVSSV_(H) CDR1 (SEQ ID NO: 104): GFTFSSYD V_(H) CDR2 (SEQ ID NO: 105):IWYDGSNK V_(H) CDR3 (SEQ ID NO: 106): ARGSGNWGFFDY3H12 VK #2 (VK 1D-16; JK1)V_(L) nucleic acid sequence (SEQ ID NO: 107)

AtgagggtcctcgctcagctcctggggctcctgctgctctgtttcccaggtgccagatgtgacatccagatgacccagtctccatcctcactgtctgcatctgtaggagacagagtcaccatcacttgtcgggcgagtcagggtattagcaggtggttagcctggtatcagcagaaaccagagaaagcccctaagtccctgatctatgctgcatccagtttgcaaagtggggtcccatcaaggttcagcggcagtggatctgggacagatttcactctcaccatcagcagcctgcagcctgaagattttgcaacttattactgccaacagtataatacttaccctcggacgttcggccaagggaccaaggtggaaatcaaaV_(L) amino acid sequence (SEQ ID NO: 108) (including signal peptide inunderlined italics):

MRVLAQLLGLLLLCFPGARC DIQMTQSPSSLSASVGDRVTITCRASQGISRWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNTYPRTFGQGTKVEIKV_(L) amino acid sequence (SEQ ID NO: 109) excluding signal peptide:

DIQMTQSPSSLSASVGDRVTITCRASQGISRWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNTYPRTF GQGTKVEIK V_(L) CDR1(SEQ ID NO: 110): QGISRW V_(L) CDR2 (SEQ ID NO: 111): AAS V_(L) CDR3(SEQ ID NO: 112): QQYNTYPRTALIGNMENTS ARE SHOWN IN FIGS. 15 AND 16

Example 10 In Vivo Non-Human Primate Study

To assess tolerance of anti-human CD27 mAb 1F5 in non-human primates, 3cynomolgus monkeys were treated with one i.v. dose of 1, 3 or 10 mg/kgof 1F5 Animals were followed for 29 days. Total lymphocytes (based onside and forward scatter size), memory B cells (CD20+ and CD95 bright),and monocytes (based on side and forward scatter size) were stained withanti-human IgG antibody (bold line) and compared to unstained controls(shaded histogram). The results are shown in FIG. 17. These results showthat the 1F5 mAb was bound to the surface of circulating lymphocytesknow to express CD27 for the entire duration of the study. Monocytes,cells that do not express CD27 did not have 1F5 binding.

In addition, to determine the effect of IF5 on circulating lymphocytepopulations, lymphocytes were stained with subset markers and the %positive cells plotted vs time in FIG. 17 for each animal treated at thedifferent doses (square points=1 mg/kg; circle points=3 mg/kg; trianglepoints=10 mg/kg). Results are shown in FIG. 18.

Collectively, the results from these studies, shown in FIGS. 17 and 18,demonstrate that 1F5 was well tolerated and did not significantlydeplete circulating lymphocytes (other than some transient depletion ofNK cells) after a single 1-10 mg/kg dose. In addition, there was noelevation in body temperatures and no detectable levels of TNF-α, IL-6or IL-1β.

Example 11 Anti-CD27 mAbs Enhanced Antigen-Specific CD8+ T-CellProliferation and Activation Pentamer Staining on Mouse Peripheral BloodCells and Splenocytes

Human CD27 transgenic mice (huCD27-Tg) were intravenously injected with5 mg of chicken ovalbumin and the panel of fully human antibodiesrecognizing human CD27 generated as in Example 1 (CD27 human mAbs). Ratanti-mouse CD27 clone AT124 and irrelevant human IgG1 were included aspositive and negative controls, respectively. Each antibody (250 μg) wasco-injected with ovalbumin on day 0 and an additional 250 μg of antibodyalone on day 1. Peripheral blood and spleen cells were harvested on day7. Splenocytes (1×10⁶) or whole blood (100 μl) were used for staining.After Fc-receptor blocking, cells were stained with 10 μl ofH-2Kb/SIINFEKL, a tetrameric complex of mouse MHC complexed with thepeptide T cell epitope from ovalbumin (Beckman Coulter), or a similarpentameric complex (Prolmmune), anti-CD8 (eBioscience) and anti-huCD27mAb or anti-msCD27 mAb (BD Biosciences) at room temperature for 30minutes. Cells were then RBC-lysed, washed and fixed, and at least100,000 events were acquired in Flow Cytometer LSR (BD Biosciences). Thefraction of tetramer- or pentamer-positive cells in the CD8⁺ or CD27⁺gated population was determined. Results are shown in FIGS. 19 and 21from which it can be seen that the anti-CD27 antibodies significantlyenhanced immune responses.

Example 12 ELISPOT Assay

Splenocytes (2.5×10⁵ and 0.5×10⁵) from the pentamer staining preparationof Example 8 above were placed on an anti-IFNγ monoclonal antibody (mAb)coated plate in triplicate wells after RBC lysis. SIINFEKL peptide wasadded at a final concentration of 2 μg/ml. A background control was setup for each sample in triplicate in the absence of peptide. Thestimulation was maintained at 37° C. in a tissue culture incubatorovernight. ELISPOT detection was performed using an ELISPOT kit (BDBiosciences) following the manufacturer's protocol. IFNγ-spot number wascounted. Results are shown in FIGS. 20 and 21, from which it can be seenthat anti-CD27 mAbs significantly enhanced T-cell activity.

Example 13 Anti-CD27 mAb Enhances T Cell Responses to a Vaccine Antigen

HuCD27 transgenic mice were immunized with 5 μg (s.c.) of theAPC-targeted vaccine comprising an anti-mouse DEC-205 IgG antibody fusedto ovalbumin (OVA) (referred to as α-mDEC-205-OVA), in combination withthe anti-CD27 human mAb 1F5 (i.p.) at various doses (25, 50, 100, 200 or400 μg). One week later splenocytes were analyzed for CD8+ T cellreactivity to the OVA SIINFEKL peptide (OVA peptide 257-264) by tetramerstaining (% tetramer positive of all CD8+ shown) and IFN-γ ELISPOT bythe procedure as generally described in Examples 8 and 9 respectively.The results are shown in FIGS. 22A-C, wherein FIG. 22A shows theprotocol used, FIG. 22B shows the results of the tetramer stainingexperiment and FIG. 22C shows the results of the IFN-gamma ELISPOTexperiment. These results indicate that the co-administered human mAb1F5 significantly enhanced the T-cell responses to the vaccine componentadministered.

Example 14 Synergistic Effect of Anti-CD27 mAb (1F5) and TLR Agonist(Poly IC) on T Cell Responses to Vaccine (Anti-DEC205-OVA)

huCD27-Tg (transgenic) and wild-type (WT) mouse littermates wereintraperitoneally injected with anti-CD27 mAb 1F5 (50 μg) on day −3, andthen subcutaneously via 4 paws injected with anti-mDec205-OVA (5 μg)plus Poly IC 0, 25, 50 or 100 μg on day 0. Spleens were collected on day7 and assessed by tetramer staining, IFNγ-ELISPOT and IFNγ-ICS. Mean±SDof positive IFNγ-ICS among gated CD8 T cells from 3 mice per group werecalculated and a panel of representative Dot Plots was collected. Theresults, shown in FIGS. 23A-D, indicate that the anti-CD27 human mAbacted synergistically with TLR3 agonist Poly IC to enhance the T-cellresponses to the vaccine component administered.

Example 15 Administration of Anti-CD27 mAb Prior to Dec205-TargetedVaccine in the Absence or Presence of TLR Agonist

huCD27-Tg mice and wild type (WT) littermates were intraperitoneallyinjected with anti-CD27 mAb (50 μg) on various days related to vaccineas indicated in FIG. 28, and subcutaneously via 4 paws injected withanti-mDec205-OVA (5 μg) plus or minus TLR agonist Poly IC-LC (20 μg) onday 0. Spleens were collected on day 7 and assessed by tetramer stainingand IFNγ-ELISPOT. A representative panel of tetramer staining amonggated CD8 T cells is shown in FIGS. 24 and 25. IFNγ-ELISPOT showed asimilar pattern.

These results show that, surprisingly, when anti-CD27 antibody isadministered in combination with a vaccine in the presence or absence ofTLR agonist, T-cell activation is greater when the antibody isadministered before the vaccine, for example a day or more before thevaccine (antigen) is administered.

Example 16 Anti-CD27 mAb Combined with TCR Activation Activates T-Cellsfrom Human CD27 Transgenic Mice

To assess the T cell activation capability of 1F5 mAb, T cells werepurified from spleen of hCD27-Tg mice by negative selection with beads.Cells were labeled with CFSE and incubated with 0.2 μg/ml of anti-CD27human mAb 1F5 or isotype control for 3 days. The cross-linkinganti-human IgG was passed through an endotoxin removal column beforeuse. IFNγ-ICS and CSFE dilution among CD8 and CD4 T cells are shown inFIGS. 26 and 27. TNFa-ICS showed the same pattern as IFNg. As shown inFIGS. 26 and 27, when combined with TCR activation, 1F5 mAb effectivelyinduces proliferation and cytokine production from T cells in vitro. Thedata show that both cross-linking with anti-human IgG and T cellreceptor activation with anti-CD3 mAb were required for 1F5 inducedproliferation and cytokine production.

Example 17 Anti-CD27 mAb Enhances the Efficacy of Vaccine in a MO4Melanoma Challenge Model

To assess in vivo anti-tumor activity, huCD27-Tg and WT control, 8 miceper group, were subcutaneously inoculated with 0.3×10⁵ MO4 cells on day0. On day 5 and 12, these mice were intraperitoneally injected withanti-CD27 mAb 1F5 (50 μg); on day 8 and 15, additional doses of CD27HuMab (50 μg) and vaccinated with anti-mDec205-OVA (5 μg). Tumor growthwas measured with calipers 2 times a week. Results are shown in FIG. 24.

In a further study, huCD27-Tg and WT control, 5 mice per group, weresubcutaneously inoculated with 1×10⁵ MO4 cells on day 0. On day 5 and12, these mice were vaccinated with anti-mDec205-OVA (5 μg) plus the TLRagonist Poly IC-LC (10 μg) intraperitoneally. On days 6 and 13, micewere injected with anti-CD27 mAb 1F5 (50 μg). Tumor growth was measuredwith calipers 2 times a week as indicated in the protocol illustrated inFIG. 285A. The results, shown in FIGS. 28B, C and D, show the effect ofno treatment (FIG. 28B), vaccine treatment alone (FIG. 28C), or vaccinetreatment in combination with anti-CD27 treatment (FIG. 28D) on tumorsize in the mice as a function of the number of days post tumorinoculation. The results demonstrate that the combination treatment withanti-CD27 mAb (1F5) significantly prolonged survival of tumor challengedmice.

Example 18 1F5 Exhibits Potent Anti-Tumor Activity in a SyngeneicTransgenic Mouse Tumor Challenge Model of the BCL₁ B-Lymphoma

To assess in vivo anti-tumor activity of 1F5, groups of 9-10 hCD27transgenic mice (Balb/c background) were challenged with 10⁷ BCL₁B-lymphoma cells administered intravenously on Day 0. Animals were thentreated with 5 doses of anti-human CD27 mAb 1F5 as indicated. As shownin FIGS. 29A and 29B, significantly prolonged survival of tumorchallenged mice, in a dose dependent manner.

Example 19 Tumor Killing in Raji Xenograph SCID Mouse Model

CB.17 SCID mice (purchased from Taconic) were maintained in apathogen-free mouse facility. Lymphoma Raji cells (1×10⁵) weresubcutaneously injected into SCID mice, 4 mice per group. On day 6,these mice were treated with CD27 human mAbs via intraperitonealadministration, 0.5 mg per dose and dosed twice a week for 3 weeks.Tumor growth was measured with calipers 3 times a week. Results of tumorgrowth and Kaplan-Meier analysis are shown in FIGS. 30A and 30B, fromwhich is can be seen that the anti-CD27 mAbs significantly prolongedsurvival of the tumor challenged mice.

In a further experiment, CB.17 SCID mice (purchased from Taconic) weremaintained in a pathogen-free mouse facility. Human lymphoma Raji cells(5×10⁵) were subcutaneously injected into SCID mice on day 0, 6 mice pergroup. On day 5, these mice were treated with anti-CD27 mAb 1F5 viaintraperitoneal administration, 0.033, 0.1 or 0.3 mg per dose and dosedtwice a week for 3 weeks. Tumor growth was measured with calipers 2times a week.

The results, shown in FIG. 31A, indicate that the anti-CD27 mAb (1F5)significantly inhibited tumor growth and thus significantly prolongedsurvival of the tumor challenged mice. A Kaplan-Meier survival plot ofthe data is also provided in FIG. 31B, which shows that median survivalwas increased by at least 10 days in mice from the treated groupcompared to the control group. A further xenograft experiment wasconducted with 1G5 and results are shown in FIG. 32.

Example 20 Tumor Killing in Daudi Xenograft SCID Mouse Model

CB.17 SCID mice (purchased from Taconic) were maintained in apathogen-free mouse facility. Human lymphoma Daudi cells (1×10⁶) weresubcutaneously injected into SCID mice on day 0, 6 mice per group. Onday 5, these mice were treated with anti-CD27 human mAb 1F5 viaintraperitoneal administration, 0.033, 0.1 or 0.3 mg per dose and dosedtwice a week for 3 weeks. Tumor growth was measured with calipers 2times a week.

The results, shown in FIG. 33, indicate that the anti-CD27 mAb (1F5)significantly inhibited tumor growth (FIG. 33A) and thus significantlyprolonged survival of the tumor challenged mice (Kaplan-Meier plot inFIG. 33B).

Example 21 Anti-CD27 mAb Engineered to not Bind Fc Receptors does notEnhance T Cell Responses to a Vaccine Antigen

HuCD27 transgenic mice were immunized with 5 μg (s.c.) of APC-targetedvaccine comprising an anti-mouse DEC-205 IgG antibody fused to ovalbumin(OVA) (referred to as α-mDEC-205-OVA), in combination with the anti-CD27human mAb 1F5 (i.p.) or mAb 1F5 mutant (Fc portion mutated to prevent Fcreceptor binding) or control IgG mAb. One week later, splenocytes wereanalyzed for CD8+ T cell reactivity to the OVA SIINFEKL peptide (OVApeptide 257-264) by IFN-γ ELISPOT by the procedure as generallydescribed.

The results, shown in FIG. 34, demonstrate that the altered human mAb1F5 does not enhance the T-cell responses to the vaccine, and thus wouldbe an effective agent for blocking the CD70/CD27 pathway.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

SUMMARY OF SEQUENCE LISTING

SEQ ID NO: DESCRIPTION 1 Human CD27 (GenBank Accession No.: AAH12160.1)2 Human CD70 (GenBank Accession No.: NP_001243) 3 V_(H) 3-33 germlinesequence is provided (Genbank Accession No AAP44382) 4 V_(H) 3-7germline sequence is provided (Genbank Accession No AAP44389) 5 3H8-1B11VH nucleic acid 6 3H8-1B11 VH amino acid including signal peptide 73H8-1B11 VH “mature” amino acid excluding signal peptide 8 3H8-1B11 VHCDR1 amino acid 9 3H8-1B11 VH CDR2 amino acid 10 3H8-1B11 VH CDR3 aminoacid 11 3H8-1B11 VL #2 nucleic acid 12 3H8-1B11 VL #2 amino acidincluding signal peptide 13 3H8-1B11 VL #2 “mature” amino acid excludingsignal peptide 14 3H8-1B11 VL #2 CDR1 amino acid 15 3H8-1B11 VL #2 CDR2amino acid 16 3H8-1B11 VL #2 CDR3 amino acid 17 3H8-1B11 VL #3 nucleicacid 18 3H8-1B11 VL #3 amino acid including signal peptide 19 3H8-1B11VL #3 “mature” amino acid excluding signal peptide 20 3H8-1B11 VL #3CDR1 amino acid 21 3H8-1B11 VL #3 CDR2 amino acid 22 3H8-1B11 VL #3 CDR3amino acid 23 2C2-1A10 VH nucleic acid 24 2C2-1A10 VH amino acidincluding signal peptide 25 2C2-1A10 VH “mature” amino acid excludingsignal peptide 26 2C2-1A10 VH CDR1 amino acid 27 2C2-1A10 VH CDR2 aminoacid 28 2C2-1A10 VH CDR3 amino acid 29 2C2-1A10 VL nucleic acid 302C2-1A10 VL amino acid including signal peptide 31 2C2-1A10 VL “mature”amino acid excluding signal peptide 32 2C2-1A10 VL CDR1 amino acid 332C2-1A10 VL CDR2 amino acid 34 2C2-1A10 VL CDR3 amino acid 35 1F5-1H5 VHnucleic acid 36 1F5-1H5 VH amino acid including signal peptide 371F5-1H5 VH “mature” amino acid excluding signal peptide 38 1F5-1H5 VHCDR1 amino acid 39 1F5-1H5 VH CDR2 amino acid 40 1F5-1H5 VH CDR3 aminoacid 41 1F5-1H5 VL #2 nucleic acid 42 1F5-1H5 VL #2 amino acid includingsignal peptide 43 1F5-1H5 VL #2 “mature” amino acid excluding signalpeptide 44 1F5-1H5 VL #1 CDR1 amino acid 45 1F5-1H5 VL #2 CDR2 aminoacid 46 1F5-1H5 VL #2 CDR3 amino acid 47 1H8-B4 VH nucleic acid 481H8-B4 VH amino acid including signal peptide 49 1H8-B4 VH “mature”amino acid excluding signal peptide 50 1H8-B4 VH CDR1 amino acid 511H8-B4 VH CDR2 amino acid 52 1H8-B4 VH CDR3 amino acid 53 1H8-B4 VLnucleic acid 54 1H8-B4 VL amino acid including signal peptide 55 1H8-B4VL “mature” amino acid excluding signal peptide 56 1H8-B4 VL CDR1 aminoacid 57 1H8-B4 VL CDR2 amino acid 58 1H8-B4 VL CDR3 amino acid 591G5-1B9 VH nucleic acid 60 1G5-1B9 VH amino acid including signalpeptide 61 1G5-1B9 VH “mature” amino acid excluding signal peptide 621G5-1B9 VH CDR1 amino acid 63 1G5-1B9 VH CDR2 amino acid 64 1G5-1B9 VHCDR3 amino acid 65 1G5-1B9 VL nucleic acid 66 1G5-1B9 VL amino acidincluding signal peptide 67 1G5-1B9 VL “mature” amino acid excludingsignal peptide 68 1G5-1B9 VL CDR1 amino acid 69 1G5-1B9 VL CDR2 aminoacid 70 1G5-1B9 VL CDR3 amino acid 71 2G9-1D11 VH nucleic acid 722G9-1D11 VH amino acid including signal peptide 73 2G9-1D11 VH “mature”amino acid excluding signal peptide 74 2G9-1D11 VH CDR1 amino acid 752G9-1D11 VH CDR2 amino acid 76 2G9-1D11 VH CDR3 amino acid 77 2G9-1D11VL nucleic acid 78 2G9-1D11 VL amino acid including signal peptide 792G9-1D11 VL “mature” amino acid excluding signal peptide 80 2G9-1D11 VLCDR1 amino acid 81 2G9-1D11 VL CDR2 amino acid 82 2G9-1D11 VL CDR3 aminoacid 83 3A10-1G10 VH nucleic acid 84 3A10-1G10 VH amino acid includingsignal peptide 85 3A10-1G10 VH “mature” amino acid excluding signalpeptide 86 3A10-1G10 VH CDR1 amino acid 87 3A10-1G10 VH CDR2 amino acid88 3A10-1G10 VH CDR3 amino acid 89 3A10-1G10 VL #1 nucleic acid 903A10-1G10 VL #1 amino acid including signal peptide 91 3A10-1G10 VL #1“mature” amino acid excluding signal peptide 92 3A10-1G10 VL #1 CDR1amino acid 93 3A10-1G10 VL #1 CDR2 amino acid 94 3A10-1G10 VL #1 CDR3amino acid 95 3A10-1G10 VL #4 nucleic acid 96 3A10-1G10 VL #4 amino acidincluding signal peptide 97 3A10-1G10 VL #4 “mature” amino acidexcluding signal peptide 98 3A10-1G10 VL #4 CDR1 amino acid 99 3A10-1G10VL #4 CDR2 amino acid 100 3A10-1G10 VL #4 CDR3 amino acid 101 3H12-1E12VH nucleic acid 102 3H12-1E12 VH amino acid including signal peptide 1033H12-1E12 VH “mature” amino acid excluding signal peptide 104 3H12-1E12VH CDR1 amino acid 105 3H12-1E12 VH CDR2 amino acid 106 3H12-1E12 VHCDR3 amino acid 107 3H12-1E12 VL #2 nucleic acid 108 3H12-1E12 VL #2amino acid including signal peptide 109 3H12-1E12 VL #2 “mature” aminoacid excluding signal peptide 110 3H12-1E12 VL #2 CDR1 amino acid 1113H12-1E12 VL #2 CDR2 amino acid 112 3H12-1E12 VL #2 CDR3 amino acid 113VH CDR3 consensus 114 VL CDR3 consensus 115 VH CDR2 consensus 116 VLCDR2 consensus 117 VH CDR1 consensus 118 VL CDR1 consensus

The invention claimed is:
 1. A method for inducing or enhancing animmune response against an antigen in a subject comprising administeringto the subject a monoclonal antibody which binds to human CD27, in anamount effective to induce or enhance an immune response against anantigen, wherein the antibody comprises a heavy chain variable regionCDR1 comprising SEQ ID NO: 38; a heavy chain variable region CDR2comprising SEQ ID NO: 39; a heavy chain variable region CDR3 comprisingSEQ ID NO: 40; a light chain variable region CDR1 comprising SEQ ID NO:44; a light chain variable region CDR2 comprising SEQ ID NO: 45; and alight chain variable region CDR3 comprising SEQ ID NO:
 46. 2. The methodof claim 1, wherein the antibody comprises a heavy chain variable regioncomprising SEQ ID NO:37.
 3. The method of claim 1, wherein the antibodycomprises a light chain variable region comprising SEQ ID NO:43.
 4. Themethod of claim 1, wherein the antibody comprises a heavy chain variableregion comprising SEQ ID NO:37 and a light chain variable regioncomprising SEQ ID NO:43.
 5. The method of claim 1, wherein the immuneresponse is an antigen-specific T cell response.
 6. A method forinducing or enhancing an immune response against an antigen in a subjectcomprising administering to the subject a monoclonal antibody whichbinds to human CD27, in an amount effective to induce or enhance animmune response against an antigen, wherein the antibody comprises heavyand light chain variable region sequences having at least 95% identityto SEQ ID NOs: 37 and 43, respectively.
 7. A method for inducing orenhancing an immune response in a subject comprising administering tothe subject a monoclonal antibody which binds to human CD27, in anamount effective to induce or enhance an immune response, wherein theantibody comprises a heavy chain variable region CDR1 comprising SEQ IDNO: 38; a heavy chain variable region CDR2 comprising SEQ ID NO: 39; aheavy chain variable region CDR3 comprising SEQ ID NO: 40; a light chainvariable region CDR1 comprising SEQ ID NO: 44; a light chain variableregion CDR2 comprising SEQ ID NO: 45; and a light chain variable regionCDR3 comprising SEQ ID NO: 46.